Dark quenchers for donor-acceptor energy transfer

ABSTRACT

The present invention provides a family of dark quenchers, termed Black Hole Quenchers (“BHQs”), that are efficient quenchers of excited state energy but which are themselves substantially non-fluorescent. Also provided are methods of using the BHQs, probes incorporating the BHQs and methods of using the probes.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 11/192,705 filed Jul. 29, 2005, now U.S. Pat. No.7,109,312, issued Sep. 19, 2006, which application is a continuation ofU.S. patent application Ser. No. 09/567,863 filed May 9, 2000, now U.S.Pat. No. 7,019,129, issued Mar. 28, 2006, each of which are incorporatedherein by reference in their entirety for all purposes.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under Grant Nos.IR43GM60848-01 and 2R44GM60848-02 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

There is a continuous and expanding need for rapid, highly specificmethods of detecting and quantifying chemical, biochemical andbiological substances as analytes in research and diagnostic mixtures.Of particular value are methods for measuring small quantities ofnucleic acids, peptides, pharmaceuticals, metabolites, microorganismsand other materials of diagnostic value. Examples of such materialsinclude narcotics and poisons, drugs administered for therapeuticpurposes, hormones, pathogenic microorganisms and viruses, antibodies,and enzymes and nucleic acids, particularly those implicated in diseasestates.

The presence of a particular analyte can often be determined by bindingmethods that exploit the high degree of specificity, which characterizesmany biochemical and biological systems. Frequently used methods arebased on, for example, antigen-antibody systems, nucleic acidhybridization techniques, and protein-ligand systems. In these methods,the existence of a complex of diagnostic value is typically indicated bythe presence or absence of an observable “label” which has been attachedto one or more of the interacting materials. The specific labelingmethod chosen often dictates the usefulness and versatility of aparticular system for detecting an analyte of interest. Preferred labelsare inexpensive, safe, and capable of being attached efficiently to awide variety of chemical, biochemical, and biological materials withoutsignificantly altering the important binding characteristics of thosematerials. The label should give a highly characteristic signal, andshould be rarely, and preferably never, found in nature. The labelshould be stable and detectable in aqueous systems over periods of timeranging up to months. Detection of the label is preferably rapid,sensitive, and reproducible without the need for expensive, specializedfacilities or the need for special precautions to protect personnel.Quantification of the label is preferably relatively independent ofvariables such as temperature and the composition of the mixture to beassayed.

A wide variety of labels have been developed, each with particularadvantages and disadvantages. For example, radioactive labels are quiteversatile, and can be detected at very low concentrations, such labelsare, however, expensive, hazardous, and their use requires sophisticatedequipment and trained personnel. Thus, there is wide interest innon-radioactive labels, particularly in labels that are observable byspectrophotometric, spin resonance, and luminescence techniques, andreactive materials, such as enzymes that produce such molecules.

Labels that are detectable using fluorescence spectroscopy are ofparticular interest, because of the large number of such labels that areknown in the art. Moreover, the literature is replete with syntheses offluorescent labels that are derivatized to allow their facile attachmentto other molecules, and many such fluorescent labels are commerciallyavailable.

In addition to being directly detected, many fluorescent labels operateto quench the fluorescence of an adjacent second fluorescent label.Because of its dependence on the distance and the magnitude of theinteraction between the quencher and the fluorophore, the quenching of afluorescent species provides a sensitive probe of molecular conformationand binding, or other, interactions. An excellent example of the use offluorescent reporter quencher pairs is found in the detection andanalysis of nucleic acids.

Fluorescent nucleic acid probes are important tools for geneticanalysis, in both genomic research and development, and in clinicalmedicine. As information from the Human Genome Project accumulates, thelevel of genetic interrogation mediated by fluorescent probes willexpand enormously. One particularly useful class of fluorescent probesincludes self-quenching probes, also known as fluorescence energytransfer probes, or FET probes. The design of different probes usingthis motif may vary in detail. In an exemplary FET probe, both afluorophore and a quencher are tethered to nucleic acid. The probe isconfigured such that the fluorophore is proximate to the quencher andthe probe produces a signal only as a result of its hybridization to anintended target. Despite the limited availability of FET probes,techniques incorporating their use are rapidly displacing alternativemethods.

Probes containing a fluorophore-quencher pair have been developed fornucleic acid hybridization assays where the probe forms a hairpinstructure, i.e., where the probe hybridizes to itself to form a loopsuch that the quencher molecule is brought into proximity with thereporter molecule in the absence of a complementary nucleic acidsequence to prevent the formation of the hairpin structure (see, forexample, WO 90/03446; European Patent Application No. 0 601 889 A2).When a complementary target sequence is present, hybridization of theprobe to the complementary target sequence disrupts the hairpinstructure and causes the probe to adopt a conformation where thequencher molecule is no longer close enough to the reporter molecule toquench the reporter molecule. As a result, the probes provide anincreased fluorescence signal when hybridized to a target sequence thanwhen they are unhybridized

Assays have also been developed for detecting a selected nucleic acidsequence and for identifying the presence of a hairpin structure usingtwo separate probes, one containing a reporter molecule and the other aquencher molecule (see, Meringue, et al., Nucleic Acids Research, 22:920-928 (1994)). In these assays, the fluorescence signal of thereporter molecule decreases when hybridized to the target sequence dueto the quencher molecule being brought into proximity with the reportermolecule.

One particularly important application for probes including areporter—quencher molecule pair is their use in nucleic acidamplification reactions, such as polymerase chain reactions (PCR), todetect the presence and amplification of a target nucleic acid sequence.In general, nucleic acid amplification techniques have opened broad newapproaches to genetic testing and DNA analysis (see, for example,Arnheim et al. Ann. Rev. Biochem., 61: 131-156 (1992)). PCR, inparticular, has become a research tool of major importance withapplications in, for example, cloning, analysis of genetic expression,DNA sequencing, genetic mapping and drug discovery (see, Arnheim et al.,supra; Gilliland et al., Proc. Natl. Acad. Sci. USA, 87: 2725-2729(1990); Bevan et al., PCR Methods and Applications, 1: 222-228 (1992);Green et al., PCR Methods and Applications, 1: 77-90 (1991); Blackwellet al., Science, 250: 1104-1110 (1990)).

Commonly used methods for detecting nucleic acid amplification productsrequire that the amplified product be separated from unreacted primers.This is typically achieved either through the use of gelelectrophoresis, which separates the amplification product from theprimers on the basis of a size differential, or through theimmobilization of the product, allowing free primer to be washed away.However, a number of methods for monitoring the amplification processwithout prior separation of primer have been described. All of them arebased on FET, and none of them detect the amplified product directly.Instead, the methods detect some event related to amplification. Forthat reason, they are accompanied by problems of high background, andare not quantitative, as discussed below.

One method, described in Wang et al. (U.S. Pat. No. 5,348,853; and Anal.Chem., 67: 1197-1203 (1995)), uses an energy transfer system in whichenergy transfer occurs between two fluorophores on the probe. In thismethod, detection of the amplified molecule takes place in theamplification reaction vessel, without the need for a separation step.This method, however, does not detect the amplified product, but insteaddetects the dissociation of primer from the “energy-sink” nucleic acid.Thus, this method is dependent on detection of a decrease in emissions;a significant portion of labeled primer must be utilized in order toachieve a reliable difference between the signals before and after thereaction.

A second method detecting an amplification product without priorseparation of primer and product is the 5′-nuclease PCR assay (alsoreferred to as the TaqMan™ assay) (Holland et al., Proc. Natl. Acad.Sci. USA, 88: 7276-7280 (1991); Lee et al., Nucleic Acids Res., 21:3761-3766 (1993)). This assay detects the accumulation of a specific PCRproduct by hybridization and cleavage of a doubly labeled fluorogenicprobe (the “TaqMan” probe) during the amplification reaction. Thefluorogenic probe consists of an nucleic acid labeled with both afluorescent reporter dye and a quencher dye. During PCR, this probe iscleaved by the 5′-exonuclease activity of DNA polymerase if, and onlyif, it hybridizes to the segment being amplified. Cleavage of the probegenerates an increase in the fluorescence intensity of the reporter dye.

In the TaqMan assay, the donor and quencher are preferably located onthe 3′- and 5′-ends of the probe, because the requirement that 5′-3′hydrolysis be performed between the fluorophore and quencher may be metonly when these two moieties are not too close to each other (Lyamichevet al., Science, 260:778-783 (1993)). This requirement is a seriousdrawback of the assay as the efficiency of energy transfer decreaseswith the inverse sixth power of the distance between the reporter andquencher. Thus, if the quencher is not close enough to the reporter toachieve the most efficient quenching the background emissions from theprobe can be quite high.

Yet another method of detecting amplification products that relies onthe use of energy transfer is the “beacon probe” method described byTyagi et al. (Nature Biotech., 14:303-309 (1996)) which is also thesubject of U.S. Pat. No. 5,312,728 to Lizardi et al. This method employsnucleic acid hybridization probes that can form hairpin structures. Onone end of the hybridization probe (either the 5′- or 3′-end) there is adonor fluorophore, and on the other end, an acceptor moiety. In thismethod, the acceptor moiety is a quencher, absorbing energy from thedonor. Thus when the beacon is in the open conformation, thefluorescence of the donor fluorophore is detectable, whereas when thebeacon is in hairpin (closed) conformation, the fluorescence of thedonor fluorophore is quenched. When employed in PCR, the molecularbeacon probe, which hybridizes to one of the strands of the PCR product,is in “open conformation,” and fluorescence is detected, while thosethat remain unhybridized will not fluoresce. As a result, the amount offluorescence will increase as the amount of PCR product increases, andthus can be used as a measure of the progress of the PCR.

Certain limitations impede the application and use of FET probes, orresult in assays that are less sensitive than they could be. Foremostamong these limitations is the presence of background fluorescenceattributable to the emission of the quencher, giving the probe a higherfluorescent noise background than is desirable. An approach that hasbeen utilized to ameliorate this limitation is the use of a quencherthat is not a fluorophore (“dark quenchers”), such as derivatives of4-(dimethylamino)azobenzene (DABCYL). DABCYL is useful as a quenchingagent for a limited group of fluorophores with whose emissioncharacteristics, the absorption characteristics of DABCYL overlap. Thelimited absorption range of DABCYL restricts the utility of thiscompound by allowing the use of a limited number of fluorophores inconjunction with DABCYL. Because relatively few fluorophores can be usedwith DABCYL in FET pairs, multiplex applications, where it is desired touse two or more fluorophores with clearly resolved fluorescence emissionspectra are difficult to design using this quencher.

In view of the limitations of presently available dark quenchers andprobes, such as FET probes constructed with these quenchers, thereexists in the art a need for improved quenchers that can be incorporatedinto probes for detecting analytes rapidly, sensitively, reliably andquantitatively. Ideal quenchers would be have little to no fluorescentquenching signal, and be easily and inexpensively prepared. Moreover, aseries of quenchers having similar physical properties, but distinctspectral properties would be particularly advantageous. Quitesurprisingly, the present invention provides such quenchers, probesincorporating these quenchers and methods for using the quenchers andprobes.

SUMMARY OF THE INVENTION

The present invention provides a family of quenchers of excited stateenergy that are substantially non-fluorescent, termed “Black HoleQuenchers” (“BHQs”). The quenchers of the invention remedy many of thedeficiencies of currently utilized dark quenchers, probes assembledusing these quenchers and methods using such quenchers and probes. Thepresent invention provides a class of dark quenchers that arefunctionalized to allow their rapid attachment to probe components usingtechniques well known in the art, or modifications of such techniquesthat are well within the abilities of those of skill in the art.Moreover, the present invention provides a class of dark quenchers thatcan be engineered to have a desired light absorption profile. Theprovision of this class of dark quenchers represents a substantialimprovement in the design of probes incorporating dark quenchers andmethods using such probes.

Many of the nucleic acid probes presently used rely on an interactionbetween the fluorophore and the quencher in order to minimize thefluorescence of the probe in the absence of its hybridization to acomplementary nucleic acid. The interaction between the fluorophore andthe quencher is typically brought about by using a nucleic acid probesequence that forms a secondary structure (e.g., hairpin, loop, etc.).Requiring that a probe adopt a secondary structure significantlycomplicates the design of the probe and greatly restricts the nucleicacid sequences that can be used as components of the probes. Incontrast, nucleic acid probes using BHQs of the present invention arefound to facilitate the interaction between the quencher and thefluorophore without requiring concomitant formation of nucleic acidsecondary structure, thereby allowing a much greater diversity ofnucleic acid sequences to be used as components of fluorescent probes.

Moreover, by varying the number and identity of the members of theconjugated system of the BHQs, the spectral properties (e.g.,absorbance) of a BHQ can be “tuned” to match the spectralcharacteristics (e.g., emission) of one or more fluorophores. Forexample, as the BHQs of the present invention can be selected to have abroad range of absorbance maxima, these quenchers are uniquely suitedfor use in multiplexing applications. Furthermore, the ability to selecta BHQ having a particular spectral characteristic allows the use of BHQsin multiplexing applications using one or more distinct fluorophore incombination with one or more distinct BHQ, thereby expanding the choicesof donor-acceptor pairs that can be incorporated into probes.

Thus, in a first aspect, the present invention provides a quencher ofexcited state energy having a structure comprising at least threeradicals selected from substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl and combinations thereof, wherein at least twoof the residues are covalently linked via an exocyclic diazo bond, thequencher further comprising a reactive functional group providing alocus for conjugation of the quencher to a carrier molecule.

In a second aspect, the present invention provides a quencher of excitedstate energy having a structure according to Formula I:

wherein R¹, R² and R³ are members independently selected fromsubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl and substituted or unsubstituted unsaturated alkyl, with theproviso that at least two of R¹, R² and R³ are members selected fromsubstituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl. X, Y and Y′ are members independently selected from reactivefunctional groups; f is a number selected from 0 to 4, inclusive, suchthat when (f+s) is greater than 1, the Y′ groups are the same ordifferent; m is a number selected from 1 to 4, inclusive, such that whenm is greater than 1, the X groups are the same or different; n is anumber from 0 to 6, inclusive, such that when (n×t) is greater than 1,the Y groups are the same or different; s is a number from 0 to 6,inclusive, such that when s is greater than 1 the R³ groups are the sameor different; and t is a number from 1 to 6, inclusive, such that when tis greater than 1 the R² groups are the same or different, and when t is1 and s is 0, a member selected from R¹, R² and combinations thereof isa member selected from substituted or unsubstituted polycyclic aryl andsubstituted or unsubstituted polycyclic heteroaryl groups.

In a third aspect, the present invention provides a quencher of excitedstate energy having a structure according to Formula II:

in which, X and Y are members independently selected from reactivefunctional groups; m is a number selected from 1 to 5, inclusive, suchthat when m is greater than 1, the X groups are the same or different; nis a number selected from 0 to 5, inclusive, such that when m is greaterthan 1, the Y groups are the same or different; s is a number selectedfrom 1 to 5, inclusive, such that when s is greater than 1, the R³groups are the same or different. R¹, R², and R³ are membersindependently selected from substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, and substituted orunsubstituted unsaturated alkyl, with the proviso that at least two ofR¹, R² and R³ are members independently selected from substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl.

In each of the above-described aspects of the invention X is preferablya member selected from —COOH, —OH and —NR′R″, wherein R′ and R″ aremembers independently selected from the group consisting of H andsubstituted or unsubstituted alkyl groups.

In a fourth aspect, the invention provides compounds having a structureaccording to Formula IV:

in which, X and Y are members independently selected from reactivefunctional groups; m is a number selected from 0 to 4, inclusive, suchthat when m is greater than 1, the X groups are the same or different; cis a number selected from 0 to 4, inclusive, such that when c is greaterthan 1, the R³ groups are the same or different; v is a number from 1 to10, inclusive, more preferably from 1 to 6, inclusive and morepreferably still between 2 and 4, inclusive. When v is greater than 1,the R² groups are the same or different. R¹, R², and R³ are membersindependently selected from substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl and substituted or unsubstitutedunsaturated alkyl, with the proviso that at least two of R¹, R² and R³are members selected from substituted or unsubstituted aryl, substitutedor unsubstituted heteroaryl and combinations thereof.

In a fifth aspect, the invention provides compounds having a structureaccording to Formula V:

wherein, R⁵, R⁶ and R⁷ are members independently selected from —NR′R″,substituted or unsubstituted aryl, nitro, substituted or unsubstitutedC₁-C₆ alkyl, and substituted or unsubstituted C₁-C₆ alkoxy, wherein R′and R″ are independently selected from H and substituted orunsubstituted C₁-C₆ alkyl. X and Y are independently selected from thegroup consisting of reactive functional groups; m is a number from 1 to2, inclusive, such that when m is 2, the X groups are the same ordifferent; n is a number from 0 to 1, inclusive; a is a number from 0 to4, inclusive, such that when a is greater than 1, the R⁵ groups are thesame or different; b is a number from 0 to 4, inclusive, such that when(v×b) is greater than 1, the R⁶ groups are the same or different; c is anumber from 0 to 5, inclusive, such that when c is greater than 1, theR⁷ groups are the same or different; and v is a number from 1 to 10,inclusive, such that when v is greater than 1, the value of b on each ofthe b phenyl rings is the same or different.

In a sixth aspect, the present invention provides a quencher of excitedstate energy having a structure, which is a member selected from:

wherein, X⁵ and X⁶ are members independently selected from H,substituted or unsubstituted C₁-C₆ alkyl, —OR′, —COOR′, —NR′R″, —SH,—OP(OX³)N(X⁴)₂, in which R′ and R″ are members independently selectedfrom the group consisting of H, and alkyl or substituted alkyl, with theproviso that at least one of X⁵ and X⁶ is a reactive functional group.X³ and X⁴ are members independently selected from CN, and substituted orunsubstituted C₁-C₆ alkyl.

In an seventh aspect, the present invention provides a method fordetermining whether a sample contains an enzyme. The method includes:(a) contacting the sample with a peptide construct that includes afluorophore; (b) exciting said fluorophore; and (c) determining afluorescence property of said sample, wherein the presence of saidenzyme in said sample results in a change in said fluorescence property.

Preferred peptide constructs include: i) a fluorophore; ii) a quenchercomprising at least three residues selected from aryl, substituted aryl,heteroaryl, substituted heteroaryl and combinations thereof, wherein atleast two of the residues are covalently linked via an exocyclic diazobond; and iii) a cleavage recognition site for the enzyme. Moreover, thepeptide is preferably in a conformation allowing donor-acceptor energytransfer between the fluorophore and the quencher when the fluorophoreis excited.

In another aspect, the invention provides a method for determiningwhether a compound alters an activity of an enzyme. Preferredembodiments of this aspect of the invention include the steps recited inconnection with the above-recited aspect of the invention and furtherinclude a step (c) determining a fluorescence property of the sample,wherein said activity of said enzyme in said sample results in a changein the fluorescence property.

In an ninth aspect, the present invention provides a method fordetecting a nucleic acid target sequence. The method includes: (a)contacting the target sequence with a detector nucleic acid; (b)hybridizing the target binding sequence to the target sequence, therebyaltering the conformation of the detector nucleic acid, causing a changein a fluorescence parameter; and (c) detecting the change in thefluorescence parameter, thereby detecting the nucleic acid targetsequence.

In the methods described herein, unless otherwise noted, a preferreddetector nucleic acid includes a single-stranded target bindingsequence. The binding sequence has linked thereto: i) a fluorophore; andii) a BHQ of the invention.

In an tenth aspect, the invention provides a method for detectingamplification of a target sequence. The method includes the use of anamplification reaction including the following steps: (a) hybridizingthe target sequence and a detector nucleic acid. The detector nucleicacid includes a single-stranded target binding sequence and anintramolecularly associated secondary structure 5′ to the target bindingsequence. At least a portion of the detector sequence forms a singlestranded tail which is available for hybridization to the targetsequence; (b) extending the hybridized detector nucleic acid on thetarget sequence with a polymerase to produce a detector nucleic acidextension product and separating the detector nucleic acid extensionproduct from the target sequence; (c) hybridizing a primer to thedetector nucleic acid extension product and extending the primer withthe polymerase, thereby linearizing the intramolecularly associatedsecondary structure and producing a change in a fluorescence parameter;and (d) detecting the change in the fluorescence parameter, therebydetecting the target sequence.

In an eleventh aspect, the invention provides a method of ascertainingwhether a first nucleic acid and a second nucleic acid hybridize. Inthis method, the first nucleic acid includes a BHQ according to theinvention. The method includes: (a) contacting the first nucleic acidwith the second nucleic acid; (b) detecting an alteration in afluorescent property of a member selected from the first nucleic acid,the second nucleic acid and a combination thereof, thereby ascertainingwhether the hybridization occurs.

In a twelfth aspect, the present invention provides a method fordetermining whether a sample contains an enzyme. The method comprises:(a) contacting the sample with a peptide construct; (b) exciting thefluorophore; and (c) determining a fluorescence property of the sample,wherein the presence of the enzyme in the sample results in a change inthe fluorescence property.

Peptide constructs useful in practicing the invention include those withthe following features: i) a fluorophore; ii) a BHQ of the invention;and iii) a cleavage recognition site for the enzyme.

In an thirteenth aspect, the invention provides methods of determiningthe amount of activity of an enzyme in a sample from an organism. Themethod includes: (a) contacting a sample comprising the enzyme and thecompound with a peptide construct comprising (b) exciting thefluorophore; and (c) determining a fluorescence property of the sample,wherein the activity of the enzyme in the sample results in a change inthe fluorescence property. Peptide constructs useful in this aspect ofthe invention are substantially similar to those described immediatelyabove.

In a fourteenth aspect, the invention provides a microarray comprising aquencher of excited state energy having a structure comprising at leastthree residues selected from aryl, substituted aryl, heteroaryl,substituted heteroaryl and combinations thereof, wherein at least two ofthe residues are covalently linked via an exocyclic diazo bond. Thequencher is conjugated directly to a solid support or to a carriermolecule attached to the solid support.

In a fifteenth aspect, the invention provides a method of probing amicroarray for the presence of a compound. The method includes: (a)contacting the microarray with a probe interacting with the compound.Preferred probes include a quencher of excited state energy having astructure comprising at least three residues selected from aryl,substituted aryl, heteroaryl, substituted heteroaryl and combinationsthereof, wherein at least two of the residues are covalently linked viaan exocyclic diazo bond; and (b) detecting a difference in afluorescence property of a member selected from the probe, the compoundand combinations thereof, thereby ascertaining the presence of thecompound.

In a sixteenth aspect, the present invention provides a mixtureincluding at least a first carrier molecule and a second carriermolecule. The first carrier molecule has covalently bound thereto afirst quencher of excited state energy having a structure comprising atleast three residues selected from aryl, substituted aryl, heteroaryl,substituted heteroaryl and combinations thereof, wherein at least two ofsaid residues are covalently linked via an exocyclic diazo bond. Thesecond carrier molecule has covalently bound thereto a second quencherof excited state energy having a structure comprising at least threeresidues selected from aryl, substituted aryl, heteroaryl, substitutedheteroaryl and combinations thereof, wherein at least two of saidresidues are covalently linked via an exocyclic diazo bond.

Additional objects and advantages of the present invention will beapparent to those of skill in the art upon examination of the detaileddescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exemplary synthetic scheme for preparing a BHQ of theinvention.

FIG. 2 is a collection of structures of exemplary BHQs (BH1, BH2 andBH3) of the invention.

FIG. 3 is a collection of structures of an exemplary BHQ attached to acontrolled pore glass support (BH1-CPG) and intermediates along thesynthesis of BH1-CPG.

FIG. 4 is a collection of structures of an exemplary BHQ attached to acontrolled pore glass support (BH2-CPG) and intermediates along thesynthesis of BH2-CPG.

FIG. 5 is a collection of structures of an exemplary BHQ attached to acontrolled pore glass support (BH3-CPG) and intermediates along thesynthesis of BH3-CPG.

FIG. 6 is a collection of structures of activated derivatives of BH1.

FIG. 7( a-b) is a comparison of the absorbance spectra of Dabcyl and theBHQs of the invention:

-   -   (a) is an overlay plot of the absorbance spectra of BH1 and the        emission spectra of three commonly used fluorophores FAM, TET        and JOE;    -   (b) is an overlay plot of the absorbance spectra of DABCYL and        the emission spectra of three commonly used fluorophores FAM,        TET and JOE.

FIG. 8 is a series of nucleic acid structures incorporating exemplaryBHQs of the invention.

DETAILED DESCRIPTION OF THE INVENTION AND THE PREFERRED EMBODIMENTS

Abbreviations

“BHQ,” as used herein, refers to “Black Hole Quenchers.”

“FET,” as used herein, refers to “Fluorescence Energy Transfer.” “FRET,”as used herein, refers to “Fluorescence Resonance Energy Transfer.”These terms are used herein to refer to both radiative and non-radiativeenergy transfer processes. For example, processes in which a photon isemitted and those involving long range electron transfer are includedwithin these terms. Throughout this specification, both of thesephenomena are subsumed under the general term “donor-acceptor energytransfer.”

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry and nucleic acidchemistry and hybridization described below are those well known andcommonly employed in the art. Standard techniques are used for nucleicacid and peptide synthesis. Generally, enzymatic reactions andpurification steps are performed according to the manufacturer'sspecifications. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (see generally, Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., which is incorporated herein by reference),which are provided throughout this document. The nomenclature usedherein and the laboratory procedures in analytical chemistry, andorganic synthetic described below are those well known and commonlyemployed in the art. Standard techniques, or modifications thereof, areused for chemical syntheses and chemical analyses.

“Analyte”, as used herein means any compound or molecule of interest forwhich a diagnostic test is performed. An analyte can be, for example, aprotein, peptide, carbohydrate, polysaccharide, glycoprotein, hormone,receptor, antigen, antibody, virus, substrate, metabolite, transitionstate analog, cofactor, inhibitor, drug, dye, nutrient, growth factor,etc., without limitation.

As used herein, “energy transfer” refers to the process by which theexcited state energy of an excited group is altered by a modifyinggroup, such as a quencher. If the excited state energy-modifying groupis a quenching group, then the fluorescence emission from thefluorescent group is attenuated (quenched). Energy transfer can occurthrough fluorescence resonance energy transfer, or through direct energytransfer. The exact energy transfer mechanisms in these two cases aredifferent. It is to be understood that any reference to energy transferin the instant application encompasses all of thesemechanistically-distinct phenomena.

As used herein, “energy transfer pair” refers to any two molecules thatparticipate in energy transfer. Typically, one of the molecules acts asa fluorescent group, and the other acts as a fluorescence-modifyinggroup. The preferred energy transfer pair of the instant inventioncomprises a fluorescent group and a quenching group of the invention.There is no limitation on the identity of the individual members of theenergy transfer pair in this application. All that is required is thatthe spectroscopic properties of the energy transfer pair as a wholechange in some measurable way if the distance between the individualmembers is altered by some critical amount. “Energy transfer pair” isused to refer to a group of molecules that form a complex within whichenergy transfer occurs. Such complexes may include, for example, twofluorescent groups, which may be different from one another and onequenching group, two quenching groups and one fluorescent group, ormultiple fluorescent groups and multiple quenching groups. In caseswhere there are multiple fluorescent groups and/or multiple quenchinggroups, the individual groups may be different from one another.

As used herein, “fluorescence-modifying group” refers to a molecule ofthe invention that can alter in any way the fluorescence emission from afluorescent group. A fluorescence-modifying group generally accomplishesthis through an energy transfer mechanism. Depending on the identity ofthe fluorescence-modifying group, the fluorescence emission can undergoa number of alterations, including, but not limited to, attenuation,complete quenching, enhancement, a shift in wavelength, a shift inpolarity, and a change in fluorescence lifetime. One example of afluorescence-modifying group is a quenching group.

As used herein, “quenching group” refers to any fluorescence-modifyinggroup of the invention that can attenuate at least partly the lightemitted by a fluorescent group. This attenuation is referred to hereinas “quenching”. Hence, illumination of the fluorescent group in thepresence of the quenching group leads to an emission signal that is lessintense than expected, or even completely absent. Quenching typicallyoccurs through energy transfer between the fluorescent group and thequenching group.

As used herein, “nucleic acid” means DNA, RNA, single-stranded,double-stranded, or more highly aggregated hybridization motifs, and anychemical modifications thereof. Modifications include, but are notlimited to, those providing chemical groups that incorporate additionalcharge, polarizability, hydrogen bonding, electrostatic interaction, andfluxionality to the nucleic acid ligand bases or to the nucleic acidligand as a whole. Such modifications include, but are not limited to,peptide nucleic acids (PNAs), phosphodiester group modifications (e.g.,phosphorothioates, methylphosphonates), 2′-position sugar modifications,5-position pyrimidine modifications, 8-position purine modifications,modifications at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo or 5-iodo-uracil; backbone modifications,methylations, unusual base-pairing combinations such as the isobases,isocytidine and isoguanidine and the like. Nucleic acids can alsoinclude non-natural bases, such as, for example, nitroindole.Modifications can also include 3′ and 5′ modifications such as cappingwith a BHQ, a fluorophore or another moiety.

“Peptide” refers to a polymer in which the monomers are amino acids andare joined together through amide bonds, alternatively referred to as apolypeptide. When the amino acids are α-amino acids, either theL-optical isomer or the D-optical isomer can be used. Additionally,unnatural amino acids, for example, β-alanine, phenylglycine andhomoarginine are also included. Commonly encountered amino acids thatare not gene-encoded may also be used in the present invention. All ofthe amino acids used in the present invention may be either the D- orL-isomer. The L-isomers are generally preferred. In addition, otherpeptidomimetics are also useful in the present invention. For a generalreview, see, Spatola, A. F., in CHEMISTRY AND BIOCHEMISTRY OF AMINOACIDS, PEPTIDES AND PROTEINS, B. Weinstein, eds., Marcel Dekker, NewYork, p. 267 (1983).

“Bioactive species,” refers to molecules that, when administered to anorganism, affect that organism. Exemplary bioactive species includepharmaceuticals, pesticides, herbicides, growth regulators and the like.Bioactive species encompasses small molecules (i.e., approximately <1000daltons), oligomers, polymers and the like. Also included are nucleicacids and their analogues, peptides and their analogues and the like.

The term “alkyl” is used herein to refer to a branched or unbranched,saturated or unsaturated, monovalent and divalent hydrocarbon radical,generally having from about 1-30 carbons and preferably, from 4-20carbons and more preferably from 6-18 carbons. When the alkyl group hasfrom 1-6 carbon atoms, it is referred to as a “lower alkyl.” Suitablealkyl radicals include, for example, structures containing one or moremethylene, methine and/or methyne groups. Branched structures have abranching motif similar to i-propyl, t-butyl, i-butyl, 2-ethylpropyl,etc. As used herein, the term encompasses “substituted alkyls,” and“cyclic alkyl.”

“Substituted alkyl” refers to alkyl as just described including one ormore substituents such as, for example, lower alkyl, aryl, acyl, halogen(i.e., alkylhalos, e.g., CF₃), hydroxy, amino, alkoxy, alkylamino,acylamino, thioamido, acyloxy, aryloxy, aryloxyalkyl, mercapto, thia,aza, oxo, both saturated and unsaturated cyclic hydrocarbons,heterocycles and the like. These groups may be attached to any carbon orsubstituent of the alkyl moiety. Additionally, these groups may bependent from, or integral to, the alkyl chain.

The term “aryl” is used herein to refer to an aromatic substituent,which may be a single aromatic ring or multiple aromatic rings which arefused together, linked covalently, or linked to a common group such as adiazo, methylene or ethylene moiety. The common linking group may alsobe a carbonyl as in benzophenone. The aromatic ring(s) may includephenyl, naphthyl, biphenyl, diphenylmethyl and benzophenone amongothers. The term “aryl” encompasses “arylalkyl” and “substituted aryl.”

“Substituted aryl” refers to aryl as just described including one ormore functional groups such as lower alkyl, acyl, halogen, alkylhalos(e.g. CF₃), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy,phenoxy, mercapto and both saturated and unsaturated cyclic hydrocarbonswhich are fused to the aromatic ring(s), linked covalently or linked toa common group such as a diazo, methylene or ethylene moiety. Thelinking group may also be a carbonyl such as in cyclohexyl phenylketone. The term “substituted aryl” encompasses “substituted arylalkyl.”

The term “arylalkyl” is used herein to refer to a subset of “aryl” inwhich the aryl group is attached to another group by an alkyl group asdefined herein.

“Substituted arylalkyl” defines a subset of “substituted aryl” whereinthe substituted aryl group is attached to another group by an alkylgroup as defined herein.

The term “acyl” is used to describe a ketone substituent, —C(O)R, whereR is alkyl or substituted alkyl, aryl or substituted aryl as definedherein.

The term “halogen” is used herein to refer to fluorine, bromine,chlorine and iodine atoms.

The term “hydroxy” is used herein to refer to the group —OH.

The term “amino” is used to —NRR′, wherein R and R′ are independently H,alkyl, aryl or substituted analogues thereof. “Amino” encompasses“alkylamino” denoting secondary and tertiary amines and “acylamino”describing the group RC(O)NR′.

The term “alkoxy” is used herein to refer to the —OR group, where R isalkyl, or a substituted analogue thereof. Suitable alkoxy radicalsinclude, for example, methoxy, ethoxy, t-butoxy, etc.

As used herein, the term “aryloxy” denotes aromatic groups that arelinked to another group directly through an oxygen atom. This termencompasses “substituted aryloxy” moieties in which the aromatic groupis substituted as described above for “substituted aryl.” Exemplaryaryloxy moieties include phenoxy, substituted phenoxy, benzyloxy,phenethyloxy, etc.

As used herein “aryloxyalkyl” defines aromatic groups attached, throughan oxygen atom to an alkyl group, as defined herein. The term“aryloxyalkyl” encompasses “substituted aryloxyalkyl” moieties in whichthe aromatic group is substituted as described for “substituted aryl.”

As used herein, the term “mercapto” defines moieties of the generalstructure —S—R wherein R is H, alkyl, aryl or heterocyclic as describedherein.

The term “saturated cyclic hydrocarbon” denotes groups such as thecyclopropyl, cyclobutyl, cyclopentyl, etc., and substituted analogues ofthese structures. These cyclic hydrocarbons can be single- or multi-ringstructures.

The term “unsaturated cyclic hydrocarbon” is used to describe amonovalent non-aromatic group with at least one double bond, such ascyclopentene, cyclohexene, etc. and substituted analogues thereof. Thesecyclic hydrocarbons can be single- or multi-ring structures.

The term “heteroaryl” as used herein refers to aromatic rings in whichone or more carbon atoms of the aromatic ring(s) are replaced by aheteroatom such as nitrogen, oxygen or sulfur. Heteroaryl refers tostructures that may be a single aromatic ring, multiple aromaticring(s), or one or more aromatic rings coupled to one or morenon-aromatic ring(s). In structures having multiple rings, the rings canbe fused together, linked covalently, or linked to a common group suchas a diazo, methylene or ethylene moiety. The common linking group mayalso be a carbonyl as in phenyl pyridyl ketone. As used herein, ringssuch as thiophene, pyridine, isoxazole, phthalimide, pyrazole, indole,furan, etc. or benzo-fused analogues of these rings are defined by theterm “heteroaryl.”

“Heteroarylalkyl” defines a subset of “heteroaryl” wherein an alkylgroup, as defined herein, links the heteroaryl group to another group.

“Substituted heteroaryl” refers to heteroaryl as just described whereinthe heteroaryl nucleus is substituted with one or more functional groupssuch as lower alkyl, acyl, halogen, alkylhalos (e.g. CF₃), hydroxy,amino, alkoxy, alkylamino, acylamino, acyloxy, mercapto, etc. Thus,substituted analogues of heteroaromatic rings such as thiophene,pyridine, isoxazole, phthalimide, pyrazole, indole, furan, etc. orbenzo-fused analogues of these rings are defined by the term“substituted heteroaryl.”

“Substituted heteroarylalkyl” refers to a subset of “substitutedheteroaryl” as described above in which an alkyl group, as definedherein, links the heteroaryl group to another group.

The term “heterocyclic” is used herein to describe a monovalentsaturated or unsaturated non-aromatic group having a single ring ormultiple condensed rings from 1-12 carbon atoms and from 1-4 heteroatomsselected from nitrogen, sulfur or oxygen within the ring. Suchheterocycles are, for example, tetrahydrofuran, morpholine, piperidine,pyrrolidine, etc.

The term “substituted heterocyclic” as used herein describes a subset of“heterocyclic” wherein the heterocycle nucleus is substituted with oneor more functional groups such as lower alkyl, acyl, halogen, alkylhalos(e.g. CF₃), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy,mercapto, etc.

The term “heterocyclicalkyl” defines a subset of “heterocyclic” whereinan alkyl group, as defined herein, links the heterocyclic group toanother group.

Introduction

The present invention provides a class of fluorescence modifiers,particularly quenchers, of excited state energy. The compounds of theinvention absorb excited state energy from a reporter fluorophore, butare themselves substantially non-fluorescent. The fluorophoretransferring the excited state energy to the quenchers of the inventionwill generally be a label that is attached to an analyte or a speciesthat interacts with, and allows detection and/or quantification of theanalyte.

Fluorescent labels have the advantage of requiring few precautions inhandling, and being amenable to high-throughput visualization techniques(optical analysis including digitization of the image for analysis in anintegrated system comprising a computer). Preferred labels are typicallycharacterized by one or more of the following: high sensitivity, highstability, low background, low environmental sensitivity and highspecificity in labeling. Many fluorescent labels are commerciallyavailable from the SIGMA chemical company (Saint Louis, Mo.), MolecularProbes (Eugene, Oreg.), R&D systems (Minneapolis, Minn.), Pharmacia LKBBiotechnology (Piscataway, N.J.), CLONTECH Laboratories, Inc. (PaloAlto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee,Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc.(Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), and Applied Biosystems (Foster City, Calif.), aswell as many other commercial sources known to one of skill.Furthermore, those of skill in the art will recognize how to select anappropriate fluorophore for a particular application and, if it notreadily available commercially, will be able to synthesize the necessaryfluorophore de novo or synthetically modify commercially availablefluorescent compounds to arrive at the desired fluorescent label.

In addition to small molecule fluorophores, naturally occurringfluorescent proteins and engineered analogues of such proteins areuseful in the present invention. Such proteins include, for example,green fluorescent proteins of cnidarians (Ward et al., Photochem.Photobiol. 35:803-808 (1982); Levine et al., Comp. Biochem. Physiol.,72B:77-85 (1982)), yellow fluorescent protein from Vibrio fischeristrain (Baldwin et al., Biochemistry 29:5509-15 (1990)),Peridinin-chlorophyll from the dinoflagellate Symbiodinium sp. (Morriset al., Plant Molecular Biology 24:673:77 (1994)), phycobiliproteinsfrom marine cyanobacteria, such as Synechococcus, e.g., phycoerythrinand phycocyanin (Wilbanks et al., J. Biol. Chem. 268:1226-35 (1993)),and the like.

The compounds, probes and methods discussed in the following sectionsare generally representative of the compositions of the invention andthe methods in which such compositions can be used. The followingdiscussion is intended as illustrative of selected aspects andembodiments of the present invention and it should not be interpreted aslimiting the scope of the present invention.

Black Hole Quenchers

The present invention provides a family of dark quenchers that arereferred to herein as “Black Hole Quenchers™” (“BHQ”s). The quenchers ofthe invention include conjugated π-bonded systems that are preferablyazo-linked aromatic species.

In a first aspect, the present invention provides a quencher of excitedstate energy having a structure comprising at least three radicalsselected from substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl and combinations thereof, wherein at least twoof the residues are covalently linked via an exocyclic diazo bond, thequencher further comprising a reactive functional group providing alocus for conjugation of the quencher to a carrier molecule. Althoughthe quenchers can be used in their free unbound form, it is generallypreferred that they be tethered to another species, thus, preferredquenchers further comprise a reactive functional group that provides alocus for conjugation of the quencher to a carrier molecule.

In another preferred embodiment, the BHQs of the invention describedherein have substantially no native fluorescence, particularly neartheir absorbance maxima or near the absorbance maxima of fluorophoresused in conjunction with the BHQs. The BHQs will preferably have anabsorbance maximum of from about 400 nm to about 760 nm, and morepreferably, of from about 500 nm to about 600 nm.

In a second aspect, the present invention provides a quencher of excitedstate energy having a structure according to Formula I:

wherein R¹, R² and R³ are members independently selected fromsubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl and substituted or unsubstituted unsaturated alkyl, with theproviso that at least two of R¹, R² and R³ are members selected fromsubstituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl. X, Y and Y′ are members independently selected from reactivefunctional groups; f is a number selected from 0 to 4, inclusive, suchthat when (f×s) is greater than 1, the Y′ groups are the same ordifferent; m is a number selected from 1 to 4, inclusive, such that whenm is greater than 1, the X groups are the same or different; n is anumber from 0 to 6, inclusive, such that when (n×t) is greater than 1,the Y groups are the same or different; s is a number from 0 to 6,inclusive, such that when s is greater than 1 the R³ groups are the sameor different; and t is a number from 1 to 6, inclusive, such that when tis greater than 1 the R² groups are the same or different, and when t is1 and s is 0, a member selected from R¹, R² and combinations thereof isa member selected from substituted or unsubstituted polycyclic aryl andsubstituted or unsubstituted polycyclic heteroaryl groups.

In a third aspect, the present invention provides a quencher of excitedstate energy having a structure according to Formula II:

in which, X and Y are members independently selected from reactivefunctional groups; m is a number selected from 1 to 5, inclusive, suchthat when m is greater than 1, the X groups are the same or different; nis a number selected from 0 to 5, inclusive, such that when m is greaterthan 1, the Y groups are the same or different; s is a number selectedfrom 1 to 5, inclusive, such that when s is greater than 1, the R³groups are the same or different. R¹, R², and R³ are membersindependently selected from substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, and substituted orunsubstituted unsaturated alkyl, with the proviso that at least two ofR¹, R² and R³ are members independently selected from substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl.

In a preferred embodiment, m is 1; n is 0; c is 1; and R¹, R² and R³ aremembers independently selected from aryl and substituted aryl.

In a preferred embodiment of each of the above-described aspects of theinvention, R¹, R² and R³ are members independently selected from aryland aryl substituted with a member selected from amino, aminoderivatives, nitro, C₁-C₆ alkyl, C₁-C₆ alkoxy and combinations thereof,and still more preferably, R¹ includes a structure according to FormulaIII:

wherein R⁴is a member selected from alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl and substituted heteroaryl.

In a fourth aspect, the invention provides compounds having a structureaccording to Formula IV:

in which, X and Y are members independently selected from reactivefunctional groups; m is a number selected from 0 to 4, inclusive, suchthat when m is greater than 1, the X groups are the same or different; cis a number selected from 0 to 4, inclusive, such that when c is greaterthan 1, the R³ groups are the same or different; v is a number from 1 to10, inclusive, more preferably from 1 to 6, inclusive and morepreferably still between 2 and 4, inclusive. When v is greater than onethe R² groups are the same or different. R¹, R², and R³ are membersindependently selected from substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl and substituted or unsubstitutedunsaturated alkyl, with the proviso that at least two of R¹, R² and R³are members selected from substituted or unsubstituted aryl, substitutedor unsubstituted heteroaryl and combinations thereof.

In a fifth aspect, the invention provides compounds having a structureaccording to Formula V:

wherein, R⁵, R⁶ and R⁷ are members independently selected from —NR′R″,substituted or unsubstituted aryl, nitro, substituted or unsubstitutedC₁-C₆ alkyl, and substituted or unsubstituted C₁-C₆ alkoxy, wherein R′and R″ are independently selected from H and substituted orunsubstituted C₁-C₆ alkyl. X and Y are independently selected from thegroup consisting of reactive functional groups; n is a number from 0 to1, inclusive; a is a number from 0 to 4, inclusive, such that when a isgreater than 1, the R⁵ groups are the same or different; b is a numberfrom 0 to 4, inclusive, such that when (v×b) is greater than 1, the R⁶groups are the same or different; c is a number from 0 to 5, inclusive,such that when c is greater than 1, the R⁷ groups are the same ordifferent; and v is a number from 1 to 10, inclusive, such that when vis greater than 1, the value of b on each of the v phenyl rings is thesame or different.

In a preferred embodiment, the present invention provides a quencher ofexcited state energy having a structure according to Formula VI:

wherein, R⁵, R⁶ and R⁷ are members independently selected from amine,alkyl amine, substituted or unsubstituted aryl, nitro, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted C₁-C₆ alkoxy; ais a number between 0 and 5, inclusive, such that when a is greater than1, the R⁵ groups are the same or different; b is a number between 0 and4, inclusive, such that when b is greater than 1, the R⁶ groups are thesame or different; c is a number between 0 and 4, inclusive, such thatwhen c is greater than 1, the R⁷ groups are the same or different; andX¹ and X² are members independently selected from C₁-C₆ alkyl or C₁-C₆substituted alkyl, —OH, —COOH, —NR′R″, —SH, —OP(OX³)(NR′R″), in which R′and R″ are members independently selected from the group consisting ofH, and alkyl or substituted alkyl.

In a sixth aspect, the present invention provides a quencher of excitedstate energy having a structure, which is a member selected from:

X⁵ and X⁶ are members independently selected from H, substituted orunsubstituted C₁-C₆ alkyl, —OR′, —COOR′, —NR′R″, —SH, —OP(OX³)N(X⁴)₂, inwhich R′ and R″ are members independently selected from the groupconsisting of H, and alkyl or substituted alkyl. At least one of X⁵ andX⁶ is a reactive functional group. X³ and X⁴ are members independentlyselected from CN, and substituted or unsubstituted C₁-C₆ alkyl.

The following discussion is generally relevant to the identity of thereactive groups of the compounds of the invention and is particularlyrelevant to the groups X, X¹ and X² in each of the above-describedaspects of the invention.

In a preferred embodiment, X is a member selected from amine, alkylamine, substituted alkyl amine, and aryl amine groups, more preferably Xhas a structure according to Formula VII:

wherein, R⁹ and R¹⁰ are members independently selected from alkyl andsubstituted alkyl; and X¹ and X² are members independently selected from—CH₃, —OH, —COOH, —NH₂, —SH, —OP(OX³)N(X⁴)₂, wherein, X³ and X⁴ aremembers independently selected from alkyl and substituted alkyl, andpreferably X³ is cyanoethyl; and X⁴ is isopropyl.

In another preferred embodiment, a member selected from R⁹, R¹⁰ andcombinations thereof comprises a polyether. Preferred polyethersinclude, for example, poly(ethylene glycol), poly(propyleneglycol) andcopolymers thereof.

In a further preferred embodiment, X has a structure according toFormula VIII:

wherein, X¹, X², X³ and X⁴ are substantially as described above and pand q are numbers independently selected from 1 to 20, inclusive,preferably from 2 to 16, inclusive.

The compounds of the invention can be prepared as a single isomer or amixture of isomers, including, for example cis-isomers, trans-isomers,diastereomers and stereoisomers. In a preferred embodiment, thecompounds are prepared as substantially a single isomer. Isomericallypure compounds are prepared by using synthetic intermediates that areisomerically pure in combination with reactions that either leave thestereochemistry at a chiral center unchanged or result in its completeinversion. Alternatively, the final product or intermediates along thesynthetic route can be resolved into a single isomer. Techniques forinverting or leaving unchanged a particular stereocenter, and those forresolving mixtures of stereoisomers are well known in the art and it iswell within the ability of one of skill in the art to choose anappropriate resolution or synthetic method for a particular situation.See, generally, Furniss et al. (eds.), VOGEL's ENCYCLOPEDIA OF PRACTICALORGANIC CHEMISTRY 5^(TH) ED., Longman Scientific and Technical Ltd.,Essex, 1991, pp. 809-816; and Heller, Acc. Chem. Res. 23: 128 (1990).

Reactive Functional Groups

The compounds of the invention bear a reactive functional group, whichcan be located at any position on an aryl nucleus or on a chain, such asan alkyl chain, attached to an aryl nucleus. When the reactive group isattached to an alkyl, or substituted alkyl chain tethered to an arylnucleus, the reactive group is preferably located at a terminal positionof an alkyl chain. Reactive groups and classes of reactions useful inpracticing the present invention are generally those that are well knownin the art of bioconjugate chemistry. Currently favored classes ofreactions available with reactive BHQs are those which proceed underrelatively mild conditions. These include, but are not limited tonucleophilic substitutions (e.g., reactions of amines and alcohols withacyl halides, active esters), electrophilic substitutions (e.g., enaminereactions) and additions to carbon-carbon and carbon-heteroatom multiplebonds (e.g., Michael reaction, Diels-Alder addition). These and otheruseful reactions are discussed in, for example, March, ADVANCED ORGANICCHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson,BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney etal., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198,American Chemical Society, Washington, D.C., 1982.

Useful reactive functional groups include, for example:

-   -   (a) carboxyl groups and various derivatives thereof including,        but not limited to, N-hydroxysuccinimide esters,        N-hydroxybenztriazole esters, acid halides, acyl imidazoles,        thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and        aromatic esters;    -   (b) hydroxyl groups, which can be converted to esters, ethers,        aldehydes, etc.    -   (c) haloalkyl groups, wherein the halide can be later displaced        with a nucleophilic group such as, for example, an amine, a        carboxylate anion, thiol anion, carbanion, or an alkoxide ion,        thereby resulting in the covalent attachment of a new group at        the site of the halogen atom;    -   (d) dienophile groups, which are capable of participating in        Diels-Alder reactions such as, for example, maleimido groups;    -   (e) aldehyde or ketone groups, such that subsequent        derivatization is possible via formation of carbonyl derivatives        such as, for example, imines, hydrazones, semicarbazones or        oximes, or via such mechanisms as Grignard addition or        alkyllithium addition;    -   (f) sulfonyl halide groups for subsequent reaction with amines,        for example, to form sulfonamides;    -   (g) thiol groups, which can be, for example, converted to        disulfides or reacted with acyl halides;    -   (h) amine or sulfhydryl groups, which can be, for example,        acylated, alkylated or oxidized;    -   (i) alkenes, which can undergo, for example, cycloadditions,        acylation, Michael addition, etc;    -   (j) epoxides, which can react with, for example, amines and        hydroxyl compounds; and    -   (k) phosphoramidites and other standard functional groups useful        in nucleic acid synthesis.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the reactions necessary to assemblethe reactive BHQ analogue. Alternatively, a reactive functional groupcan be protected from participating in the reaction by the presence of aprotecting group. Those of skill in the art understand how to protect aparticular functional group such that it does not interfere with achosen set of reaction conditions. For examples of useful protectinggroups, see, for example, Greene et al., PROTECTIVE GROUPS IN ORGANICSYNTHESIS, John Wiley & Sons, New York, 1991.

Donor and Acceptor Moieties

One of the advantages of the compounds of the invention is that a widerange of energy donor molecules can be used in conjunction with theBHQs. A vast array of fluorophores are known to those of skill in theart. See, for example, Cardullo et al., Proc. Natl. Acad. Sci. USA 85:8790-8794 (1988); Dexter, D. L., J. of Chemical Physics 21: 836-850(1953); Hochstrasser et al., Biophysical Chemistry 45: 133-141 (1992);Selvin, P., Methods in Enzymology 246: 300-334 (1995); Steinberg, I.Ann. Rev. Biochem., 40: 83-114 (1971); Stryer, L. Ann. Rev. Biochem.,47: 819-846 (1978); Wang et al., Tetrahedron Letters 31: 6493-6496(1990); Wang et al., Anal. Chem. 67: 1197-1203 (1995).

A non-limiting list of exemplary donors that can be used in conjunctionwith the quenchers of the invention is provided in Table 1.

TABLE 1 Suitable moieties that can be selected as donors or acceptors indonor-acceptor energy transfer pairs4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid acridine andderivatives: acridine acridine isothiocyanate5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS)4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonateN-(4-aniIino-1-naphthyl)maleimide anthranilamide BODIPY Brilliant Yellowcoumarin and derivatives: coumarin 7-amino-4-methylcoumarin (AMC,Coumarin 120) 7-amino-4-trifluoromethylcouluarin (Coumaran 151) cyaninedyes cyanosine 4′,6-diaminidino-2-phenylindole (DAPI)5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red)7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarindiethylenetriamine pentaacetate4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid5-[dimethylamino]naphthalene-l-sulfonyl chloride (DNS, dansylchloride)4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL)4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC) eosin andderivatives: eosin eosin isothiocyanate erythrosin and derivatives:erythrosin B erythrosin isothiocyanate ethidium fluorescein andderivatives: 5-carboxyfluorescein (FAM)5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF)2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE) fluoresceinfluorescein isothiocyanate QFITC (XRITC) fluorescamine IR144 IR1446Malachite Green isothiocyanate 4-methylumbelliferone orthocresolphthalein nitrotyrosine pararosaniline Phenol Red B-phycoerythrino-phthaldialdehyde pyrene and derivatives: pyrene pyrene butyratesuccinimidyl 1-pyrene butyrate quantum dots Reactive Red 4 (Cibacron ™Brilliant Red 3B-A) rhodamine and derivatives: 6-carboxy-X-rhodamine(ROX) 6-carboxyrhodamine (R6G) lissamine rhodamine B sulfonyl chloriderhodamine (Rhod) rhodamine B rhodamine 123 rhodamine X isothiocyanatesulforhodamine B sulforhodamine 101 sulfonyl chloride derivative ofsulforhodamine 101 (Texas Red) N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA) tetramethyl rhodamine tetramethyl rhodamine isothiocyanate(TRITC) riboflavin rosolic acid terbium chelate derivatives

There is a great deal of practical guidance available in the literaturefor selecting appropriate donor-acceptor pairs for particular probes, asexemplified by the following references: Pesce et al., Eds.,FLUORESCENCE SPECTROSCOPY (Marcel Dekker, New York, 1971); White et al.,FLUORESCENCE ANALYSIS: A PRACTICAL APPROACH (Marcel Dekker, New York,1970); and the like. The literature also includes references providingexhaustive lists of fluorescent and chromogenic molecules and theirrelevant optical properties for choosing reporter-quencher pairs (see,for example, Berlman, HANDBOOK OF FLUORESCENCE SPECTRA OF AROMATICMOLECULES, 2nd Edition (Academic Press, New York, 1971); Griffiths,COLOUR AND CONSTITUTION OF ORGANIC MOLECULES (Academic Press, New York,1976); Bishop, Ed., INDICATORS (Pergamon Press, Oxford, 1972); Haugland,HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (Molecular Probes,Eugene, 1992) Pringsheim, FLUORESCENCE AND PHOSPHORESCENCE (IntersciencePublishers, New York, 1949); and the like. Further, there is extensiveguidance in the literature for derivatizing reporter and quenchermolecules for covalent attachment via common reactive groups that can beadded to a nucleic acid, as exemplified by the following references:Haugland (supra); Ullman et al., U.S. Pat. No. 3,996,345; Khanna et al.,U.S. Pat. No. 4,351,760. Thus, it is well within the abilities of thoseof skill in the art to choose an energy exchange pair for a particularapplication and to conjugate the members of this pair to a probemolecule, such as, for example, a nucleic acid, peptide or otherpolymer.

Generally, it is preferred that an absorbance band of the BHQsubstantially overlap the fluorescence emission band of the donor. Whenthe donor (fluorophore) is a component of a probe that utilizesdonor-acceptor energy transfer, the donor fluorescent moiety and thequencher (acceptor) of the invention are preferably selected so that thedonor and acceptor moieties exhibit donor-acceptor energy transfer whenthe donor moiety is excited. One factor to be considered in choosing thefluorophore-quencher pair is the efficiency of donor-acceptor energytransfer between them. Preferably, the efficiency of FRET between thedonor and acceptor moieties is at least 10%, more preferably at least50% and even more preferably at least 80%. The efficiency of FRET caneasily be empirically tested using the methods both described herein andknown in the art.

The efficiency of energy transfer between the donor-acceptor pair canalso be adjusted by changing the ability of the donor and acceptorgroups to dimerize or closely associate. If the donor and acceptormoieties are known or determined to closely associate, an increase ordecrease in association can be promoted by adjusting the length of alinker moiety, or of the probe itself, between the donor and acceptor.The ability of donor-acceptor pair to associate can be increased ordecreased by tuning the hydrophobic or ionic interactions, or the stericrepulsions in the probe construct. Thus, intramolecular interactionsresponsible for the association of the donor-acceptor pair can beenhanced or attenuated. Thus, for example, the association between thedonor-acceptor pair can be increased by, for example, utilizing a donorbearing an overall negative charge and an acceptor with an overallpositive charge.

In addition to fluorophores that are attached directly to a probe, thefluorophores can also be attached by indirect means. In this embodiment,a ligand molecule (e.g., biotin) is generally covalently bound to theprobe species. The ligand then binds to another molecules (e.g.,streptavidin) molecule, which is either inherently detectable orcovalently bound to a signal system, such as a fluorescent compound, oran enzyme that produces a fluorescent compound by conversion of anon-fluorescent compound. Useful enzymes of interest as labels include,for example, hydrolases, particularly phosphatases, esterases andglycosidases, hydrolases, peptidases or oxidases, particularlyperoxidases, and. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.,as discussed above. For a review of various labeling or signal producingsystems that can be used, see, U.S. Pat. No. 4,391,904.

Presently preferred donors of use in conjunction with BHQ, include, forexample, xanthene dyes, including fluoresceins, cyanine dyes andrhodamine dyes. Many suitable forms of these compounds are widelyavailable commercially with substituents on their phenyl moieties, whichcan be used as the site for bonding or as the bonding functionality forattachment to an nucleic acid. Another group of preferred fluorescentcompounds are the naphthylamines, having an amino group in the alpha orbeta position. Included among such naphthylamino compounds are1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonateand 2-p-touidinyl-6-naphthalene sulfonate. Other donors include3-phenyl-7-isocyanatocoumarin, acridines, such as9-isothiocyanatoacridine and acridine orange;N-(p-(2-benzoxazolyl)phenyl)maleimide; benzoxadiazoles, stilbenes,pyrenes, and the like.

For clarity of illustration, the discussion below focuses on attachingBHQs and fluorophores to nucleic acids. The focus on nucleic acid probesis not intended to limit the scope of probe molecules to which BHQs canbe attached. Those of skill in the art will appreciate that BHQs canalso be attached to small molecules, proteins, peptides, syntheticpolymers, solid supports and the like using standard synthetic chemisty.

In a presently preferred embodiment, in which the probe is a nucleicacid probe, the reporter molecule is a fluorescein dye (FAM). Thefluorescein moiety is preferably attached to either the 3′- or the5′-terminus of the nucleic acid, although internal sites are alsoaccessible and have utility for selected purposes. Whichever terminusthe FAM derivative is attached to, the BHQ will generally be attached toits antipode, or at a position internal to the nucleic acid chain. TheFAM donor is preferably introduced using a 6-FAM amidite. Differentdonor groups are also preferably introduced using an amidite derivativeof the donor. Alternatively, donor groups comprising reactive groups(e.g., isothiocyanates, active esters, etc.) can be introduced viareaction with a reactive moiety on a tether or linker arm attached tothe nucleic acid (e.g., hexyl amine).

In yet another preferred embodiment, the donor moiety can be attached atthe 3′-terminus of a nucleic acid by the use of a derivatized synthesissupport. For example, TAMRA (tetramethylrhodamine carboxylic acid) isattached to a nucleic acid 3′-terminus using a solid support that isderivatized with an analogue of this fluorophore (BiosearchTechnologies, Inc.)

In view of the well-developed body of literature concerning theconjugation of small molecules to nucleic acids, many other methods ofattaching donor/acceptor pairs to nucleic acids will be apparent tothose of skill in the art. For example, rhodamine and fluorescein dyesare conveniently attached to the 5′-hydroxyl of an nucleic acid at theconclusion of solid phase synthesis by way of dyes derivatized with aphosphoramidite moiety (see, for example, Woo et al., U.S. Pat. No.5,231,191; and Hobbs, Jr., U.S. Pat. No. 4,997,928).

More specifically, there are many linking moieties and methodologies forattaching groups to the 5′- or 3′-termini of nucleic acids, asexemplified by the following references: Eckstein, editor, Nucleic acidsand Analogues: A Practical Approach (IRL Press, Oxford, 1991); Zuckermanet al., Nucleic Acids Research, 15: 5305-5321 (1987) (3′-thiol group onnucleic acid); Sharma et al., Nucleic Acids Research, 19: 3019 (1991)(3′-sulfhydryl); Giusti et al., PCR Methods and Applications, 2: 223-227(1993) and Fung et al., U.S. Pat. No. 4,757,141 (5′-phosphoamino groupvia Aminolink TM II available from P.E. Biosystems, Calif.) Stabinsky,U.S. Pat. No. 4,739,044 (3-aminoalkylphosphoryl group); Agrawal et al.,Tetrahedron Letters, 31: 1543-1546 (1990) (attachment viaphosphoramidate linkages); Sproat et al., Nucleic Acids Research, 15:4837 (1987) (5-mercapto group); Nelson et al., Nucleic Acids Research,17: 7187-7194 (1989) (3′-amino group), and the like.

Means of detecting fluorescent labels are well known to those of skillin the art. Thus, for example, fluorescent labels can be detected byexciting the fluorophore with the appropriate wavelength of light anddetecting the resulting fluorescence. The fluorescence can be detectedvisually, by means of photographic film, by the use of electronicdetectors such as charge coupled devices (CCDs) or photomultipliers andthe like. Similarly, enzymatic labels may be detected by providing theappropriate substrates for the enzyme and detecting the resultingreaction product.

Synthesis

The compounds of the invention are synthesized by an appropriatecombination of generally well-known synthetic methods. Techniques usefulin synthesizing the compounds of the invention are both readily apparentand accessible to those of skill in the relevant art. The discussionbelow is offered to illustrate certain of the diverse methods availablefor use in assembling the compounds of the invention, it is not intendedto define the scope of reactions or reaction sequences that are usefulin preparing the compounds of the present invention.

One method of synthesizing compounds of the invention is set forth inScheme 1(FIG. 1). Scheme 1 is a generalized schematic of one syntheticscheme useful with the BHQs of the invention. An azido derivative of adye is coupled to an aryl derivative 1, at pH 9, forming thecorresponding diazo adduct 2. The diol 2 is monoprotected with a group,such as the dimethoxytrityl group to form compound 3, having one freehydroxyl moiety. Compound 3 is converted to phosphoramidite 4 bycontacting it with an agent, such as 2-cyanoethyldiisopropylchlorophosphoramidite in the presence of a mildly acidicactivator, such as tetrazole. The phosphoramidite is coupled to ahydroxyl-bearing controlled pore glass support and subsequently oxidizedto the corresponding phosphotriester derivative, thereby forming anappropriate starting material, 5, for the synthesis of an array ofnucleic acids derivatized at the 3′-position with a BHQ.

The above-recited synthetic scheme is intended to be exemplary of oneembodiment of the invention, those of skill in the art will recognizethat many other synthetic strategies employing reactive BHQ analoguesare available. For example, by a slight modification of the methodabove, a derivative appropriate for modification of a nucleic acid atthe 5′-position is easily accessible. In an alternative scheme, compound4, is not tethered to a solid support, but is added as the final subunitduring nucleic acid synthesis, to prepare a nucleic acid with a 5′-BHQgroup.

The above-described synthetic scheme can be practiced using a variety ofBHQ compounds of the invention, such as those set forth in FIG. 2. FIG.2 provides the structures of three exemplary BHQs, BH1 (6), BH2 (10) andBH3 (14).

FIG. 3 sets forth the structures of a phosphoramidite (8) of BH1 (7),and a derivative of BH1, which is tethered to a controlled pore glasssupport (9). Both the phosphoramidite and the CPG conjugate can beprepared by art-recognized methods, including those set forth herein.Similarly, FIG. 4 sets forth the structures of a phosphoramidite (12) ofBH2 (11), and a derivative of BH2, which is tethered to a controlledpore glass support (13) and FIG. 5 sets forth the structures of aphosphoramidite (16) of BH1 (15), and a derivative of BH3, which istethered to a controlled pore glass support (17).

In a still further modification of the scheme of FIG. 1, compound 4 iscoupled to a nucleic acid intermediate between the 3′- and 5′-positions,the DMT group is removed using standard nucleic acid chemistry, or amodification thereof, and a nucleic acid subunit is tethered to thedeprotected primary hydroxyl group as though the hydroxyl group were the5′-hydroxyl of a preceding nucleic acid subunit, thereby providing anucleic acid having a BHQ moiety at an internal position.

Assays and BHQ-Bearing Probes

In another preferred embodiment, the present invention provides a BHQthat is tethered to another molecule, such as a probe molecule andassays using these probes.

Assays

The following discussion is generally relevant to the assays describedherein. This discussion is intended to illustrate the invention byreference to certain preferred embodiments and should not be interpretedas limiting the scope of probes and assay types in which the compoundsof the invention find use. Other assay formats utilizing the compoundsof the invention will be apparent to those of skill in the art.

In general, to determine the concentration of a target molecule, suchas, for example, a nucleic acid, it is preferable to first obtainreference data in which constant amounts of probe and nucleic acidligand are contacted with varying amounts of target. The fluorescenceemission of each of the reference mixtures is used to derive a graph ortable in which target concentration is compared to fluorescenceemission. For example, a probe that: a) hybridizes to a target-freenucleic acid ligand; and b) has a stem-loop architecture with the 5′ and3′ termini being the sites of fluorescent group and BHQ labeling, can beused to obtain such reference data. Such a probe gives a characteristicemission profile in which the fluorescence emission decreases as thetarget concentration increases in the presence of a constant amount ofprobe and nucleic acid ligand. Then, a test mixture with an unknownamount of target is contacted with the same amount of first nucleic acidligand and second probe, and the fluorescence emission is determined.The value of the fluorescence emission is then compared with thereference data to obtain the concentration of the target in the testmixture.

Multiplex Analyses

In another preferred embodiment, the quenchers of the invention areutilized as a component of one or more probes used in a multiplex assayfor detecting one or more species in a mixture.

Probes that include the BHQs of the invention are particularly useful inperforming multiplex-type analyses and assays. In a typical multiplexanalysis, two or more distinct species (or regions of one or morespecies) are detected using two or more probes, wherein each of theprobes is labeled with a different fluorophore. Preferred species usedin multiplex analyses relying on donor-acceptor energy transfer meet atleast two criteria: the fluorescent species is bright and spectrallywell-resolved; and the energy transfer between the fluorescent speciesand the quencher is efficient.

Thus, in a further embodiment, the invention provides a mixturecomprising at least a first carrier molecule and a second carriermolecule. The first carrier molecule has covalently bound thereto afirst quencher of excited state energy having a structure comprising atleast three radicals selected from aryl, substituted aryl, heteroaryl,substituted heteroaryl and combinations thereof. At least two of theradicals are covalently linked via an exocyclic diazo bond. The mixturealso includes a second carrier molecule. The second carrier molecule hascovalently bound thereto a second quencher of excited state energyhaving a structure comprising at least three radicals selected fromaryl, substituted aryl, heteroaryl, substituted heteroaryl andcombinations thereof, wherein at least two of the radicals arecovalently linked via an exocyclic diazo bond.

The BHQs of the invention allow for the design of multiplex assays inwhich more than one quencher structure is used in the assay. A number ofdifferent multiplex assays using the BHQs of the invention will beapparent to one of skill in the art. In one exemplary assay, each of theat least two distinct BHQ quenchers is used to quench energy derivedfrom one or more identical fluorophore. Alternatively, an assay can bepracticed in which each distinct BHQ quenches energy derived from adistinct fluorophore to which the BHQ is “matched.” The fluorophores canbe bound to the same molecule as the BHQ or to a different molecule.Moreover, similar to the BHQs and the fluorophores, the carriermolecules of use in a particular assay system can be the same ordifferent.

In addition to the mixtures described above, the present invention alsoprovides a method for detecting or quantifying a particular molecularspecies. The method includes: (a) contacting the species with a mixturesuch as that described above; and (b) detecting a change in afluorescent property of one or more component of the mixture, themolecular species or a combination thereof, thereby detecting orquantifying the molecular species.

Because of the ready availability of BHQs of the invention havingdifferent absorbance characteristics, the compounds of the invention areparticularly well suited for use in multiplex applications. Access toBHQs having a range of absorbance characteristics allows for the designof donor-acceptor energy transfer probes in which the acceptor emissionproperties and the BHQ absorbance properties are substantially matched,thereby providing a useful level of spectral overlap (see, for example,FIG. 7).

The simultaneous use of two or more probes using donor-acceptor energytransfer is known in the art. For example, multiplex assays usingnucleic acid probes with different sequence specificities have beendescribed. Fluorescent probes have been used to determine whether anindividual is homozygous wild-type, homozygous mutant or heterozygousfor a particular mutation. For example, using one quenched-fluoresceinmolecular beacon that recognizes the wild-type sequence and anotherrhodamine-quenched molecular beacon that recognizes a mutant allele, itis possible to genotype individuals for the β-chemokine receptor(Kostrikis et al. Science 279:1228-1229 (1998)). The presence of only afluorescein signal indicates that the individual is wild-type, and thepresence of rhodamine signal only indicates that the individual is ahomozygous mutant. The presence of both rhodamine and fluorescein signalis diagnostic of a heterozygote. Tyagi et al. Nature Biotechnology 16:49-53 (1998)) have described the simultaneous use of four differentlylabeled molecular beacons for allele discrimination, and Lee et al., BioTechniques 27: 342-349 (1999) have described seven color homogenousdetection of six PCR products.

The quenchers of the present invention can be used in multiplex assaysdesigned to detect and/or quantify substantially any species, including,for example, whole cells, viruses, proteins (e.g., enzymes, antibodies,receptors), glycoproteins, lipoproteins, subcellular particles,organisms (e.g., Salmonella), nucleic acids (e.g., DNA, RNA, andanalogues thereof), polysaccharides, lipopolysaccharides, lipids, fattyacids, non-biological polymers and small molecules (e.g., toxins, drugs,pesticides, metabolites, hormones, alkaloids, steroids).

Probes

The invention provides probes including BHQ moieties conjugated to, forexample, a target species (e.g., receptor, enzyme, etc.) a ligand for atarget species (e.g., nucleic acid, peptide, etc.), a small molecule(e.g., drug, pesticide, etc.), and the like. The probes can be used forin vitro and in vivo applications.

A particularly unexpected and surprising advantage of the BHQs is theirability to quench excited state energy from fluorophores attached tocarrier molecules (e.g., nucleic acids) without the need to designsecondary structure forming components (e.g., hairpins, loops, etc.)into the carrier molecule to bring the fluorophore and the BHQ intoproximity. The energy transfer pairs of probes presently used in the arttypically require the introduction of some form of secondary structurein order to function properly, thereby seriously constraining theidentity of species that can be used as carrier molecules. Thus, theprobes of the present invention can be of simple design, can be producedmore inexpensively and used to probe a much greater array of systems inmuch less time than current art-recognized probes.

Yet another unexpected property of the BHQs of the invention is theirrobustness under a variety of synthetic conditions used to attach theBHQs to a carrier molecule. For example, many of the BHQs of theinvention survive the conditions necessary for automated synthesis ofnucleic acids without undergoing any substantial degree of degradationor alteration. In contrast, many of the art-recognized quencherspresently in use require the use of special conditions to assemble thecarrier molecule to which they are attached, or they have to be attachedafter the completion of the carrier molecule synthesis. The additionalcomplexity of the synthesis of a probe increases both the duration ofthe synthesis and its cost.

Small Molecule Probes

The BHQs of the invention can be used as components of small moleculeprobes. In a preferred design, a small molecule probe includes afluorophore or fluorophore precursor and a BHQ. In an exemplaryembodiment, an agent, such as an enzyme cleaves the BHQ, the fluorophoreor both from the small molecule generating fluorescence in the systemunder investigation (see, for example, Zlokarnik et al., Science 279:84-88 (1998)).

Nucleic Acid Probes

The dark quenchers of the invention are useful in conjunction withnucleic-acid probes and they can be used as components of detectionagents in a variety of DNA amplification/quantification strategiesincluding, for example, 5′-nuclease assay, Strand DisplacementAmplification (SDA), Nucleic Acid Sequence-Based Amplification (NASBA),Rolling Circle Amplification (RCA), as well as for direct detection oftargets in solution phase or solid phase (e.g., array) assays.Furthermore, the BHQ-derivatized nucleic acids can be used in probes ofsubstantially any format, including, for example, format selected frommolecular beacons, Scorpion probes™, Sunrise probes™, conformationallyassisted probes, light up probes, Invader Detection probes, and TaqMan™probes. See, for example, Cardullo, R., et al., Proc. Natl. Acad. Sci.USA, 85:8790-8794 (1988); Dexter, D. L., J. Chem. Physics, 21:836-850(1953); Hochstrasser, R. A., et al., Biophysical Chemistry, 45:133-141(1992); Selvin, P., Methods in Enzymology, 246:300-334 (1995);Steinberg, I., Ann. Rev. Biochem., 40:83-114 (1971); Stryer, L., Ann.Rev. Biochem., 47:819-846 (1978); Wang, G., et al., Tetrahedron Letters,31:6493-6496 (1990); Wang, Y., et al., Anal. Chem., 67:1197-1203 (1995);Debouck, C., et al., in supplement to nature genetics, 21:48-50 (1999);Rehman, F. N., et al., Nucleic Acids Research, 27:649-655 (1999);Cooper, J. P., et al., Biochemistry, 29:9261-9268 (1990); Gibson, E. M.,et al., Genome Methods, 6:995-1001 (1996); Hochstrasser, R. A., et al.,Biophysical Chemistry, 45:133-141 (1992); Holland, P. M., et al., ProcNatl. Acad. Sci USA, 88:7276-7289 (1991); Lee, L. G., et al., NucleicAcids Rsch., 21:3761-3766 (1993); Livak, K. J., et al., PCR Methods andApplications, Cold Spring Harbor Press (1995); Vamosi, G., et al.,Biophysical Journal, 71:972-994 (1996); Wittwer, C. T., et al.,Biotechniques, 22:176-181 (1997); Wittwer, C. T., et al., Biotechniques,22:130-38 (1997); Giesendorf, B. A. J., et al., Clinical Chemistry,44:482-486 (1998); Kostrikis, L. G., et al., Science, 279:1228-1229(1998); Matsuo, T., Biochemica et Biophysica Acta, 1379:178-184 (1998);Piatek, A. S., et al., Nature Biotechnology, 16:359-363 (1998);Schofield, P., et al., Appl. Environ. Microbiology, 63:1143-1147 (1997);Tyagi S., et al., Nature Biotechnology, 16:49-53 (1998); Tyagi, S., etal., Nature Biotechnology, 14:303-308 (1996); Nazarenko, I. A., et al.,Nucleic Acids Research, 25:2516-2521 (1997); Uehara, H., et al.,Biotechniques, 26:552-558 (1999); D. Whitcombe, et al., NatureBiotechnology, 17:804-807 (1999); Lyamichev, V., et al., NatureBiotechnology, 17:292 (1999); Daubendiek, et al., Nature Biotechnology,15:273-277 (1997); Lizardi, P. M., et al., Nature Genetics, 19:225-232(1998); Walker, G., et al., Nucleic Acids Res., 20:1691-1696 (1992);Walker, G. T., et al., Clinical Chemistry, 42:9-13 (1996); and Compton,J., Nature, 350:91-92 (1991).

Thus, in a further aspect, the present invention provides a method fordetecting a nucleic acid target sequence. The method includes: (a)contacting the target sequence with a detector nucleic acid; (b)hybridizing the target binding sequence to the target sequence, therebyaltering the conformation of the detector nucleic acid, causing a changein a fluorescence parameter; and (c) detecting the change in thefluorescence parameter, thereby detecting the nucleic acid targetsequence.

In the methods described herein, unless otherwise noted, a preferreddetector nucleic acid includes a single-stranded target bindingsequence. The binding sequence has linked thereto: i) a fluorophore; andii) a BHQ of the invention. Moreover, prior to its hybridization to acomplementary sequence, the detector nucleic acid is preferably in aconformation that allows donor-acceptor energy transfer between thefluorophore and the BHQ when the fluorophore is excited. Furthermore, ineach of the methods described in this section, a change in fluorescenceis detected as an indication of the presence of the target sequence. Thechange in fluorescence is preferably detected in-real time.

Presently preferred nucleic acid probes do not require the carriermolecule to adopt a secondary structure for the probe to function.

In this method, and unless otherwise noted, the other methods describedin this section, the detector nucleic acid can assume substantially anyintramolecularly associated secondary structure, but this structure ispreferably a member selected from hairpins, stem-loop structures,pseudoknots, triple helices and conformationally assisted structures.Moreover, the intramolecularly base-paired secondary structurepreferably comprises a portion of the target binding sequence.

In another aspect, the invention provides a method for detectingamplification of a target sequence. The method includes the use of anamplification reaction including the following steps: (a) hybridizingthe target sequence and a detector nucleic acid. The detector nucleicacid includes a single-stranded target binding sequence and anintramolecularly associated secondary structure 5′ to the target bindingsequence. At least a portion of the detector sequence forms a singlestranded tail which is available for hybridization to the targetsequence; (b) extending the hybridized detector nucleic acid on thetarget sequence with a polymerase to produce a detector nucleic acidextension product and separating the detector nucleic acid extensionproduct from the target sequence; (c) hybridizing a primer to thedetector nucleic acid extension product and extending the primer withthe polymerase, thereby linearizing the intramolecularly associatedsecondary structure and producing a change in a fluorescence parameter;and (d) detecting the change in the fluorescence parameter, therebydetecting the target sequence.

In yet a further aspect, the invention provides a method of ascertainingwhether a first nucleic acid and a second nucleic acid hybridize. Inthis method, the first nucleic acid includes a BHQ according to theinvention. The method includes: (a) contacting the first nucleic acidwith the second nucleic acid; (b) detecting an alteration in afluorescent property of a member selected from the first nucleic acid,the second nucleic acid and a combination thereof, thereby ascertainingwhether the hybridization occurs.

A probe bearing both a BHQ and a fluorophore can be used or,alternatively, one or more of the nucleic acids can be singly labeledwith a BHQ or fluorophore. When a nucleic acid singly labeled with a BHQis the probe, the interaction between the first and second nucleic acidscan be detected by observing the interaction between the BHQ and thenucleic acid or, more preferably, the quenching by the BHQ of thefluorescence of a fluorophore attached to the second nucleic acid.

In addition to their general utility in probes designed to investigatenucleic acid amplification, detection and quantification, the presentdark quenchers can be used in substantially any nucleic acid probeformat now known or later discovered. For example, the dark quenchers ofthe invention can be incorporated into probe motifs, such as Taqman™probes (Held et al., Genome Res. 6: 986-994 (1996), Holland et al.,Proc. Nat. Acad. Sci. USA 88: 7276-7280 (1991), Lee et al., NucleicAcids Res. 21: 3761-3766 (1993)), molecular beacons (Tyagi et al.,Nature Biotechnology 14:303-308 (1996), Jayasena et al., U.S. Pat. No.5,989,823, issued Nov. 23, 1999)) scorpion probes (Whitcomb et al.,Nature Biotechnology 17: 804-807 (1999)), sunrise probes (Nazarenko etal., Nucleic Acids Res. 25: 2516-2521 (1997)), conformationally assistedprobes (Cook, R., copending and commonly assigned U.S. ProvisionalApplication 60/138,376, filed Jun. 9, 1999), peptide nucleic acid(PNA)-based light up probes (Kubista et al., WO 97/45539, December1997), double-strand specific DNA dyes (Higuchi et al, Bio/Technology10: 413-417 (1992), Wittwer et al, BioTechniques 22: 130-138 (1997)) andthe like. These and other probe motifs with which the present quencherscan be used are reviewed in NONISOTOPIC DNA PROBE TECHNIQUES, AcademicPress, Inc. 1992.

The nucleic acids for use in the probes of the invention can be anysuitable size, and are preferably in the range of from about 10 to about100 nucleotides, more preferably from about 10 to about 80 nucleotidesand more preferably still, from about 20 to about 40 nucleotides. Theprecise sequence and length of a nucleic acid probe of the inventiondepends in part on the nature of the target polynucleotide to which itbinds. The binding location and length may be varied to achieveappropriate annealing and melting properties for a particularembodiment. Guidance for making such design choices can be found in manyart-recognized references.

Preferably, the 3′-terminal nucleotide of the nucleic acid probe isblocked or rendered incapable of extension by a nucleic acid polymerase.Such blocking is conveniently carried out by the attachment of a donoror acceptor moiety to the terminal 3′-position of the nucleic acidprobe, either directly or by a linking moiety.

The nucleic acid can comprise DNA, RNA or chimeric mixtures orderivatives or modified versions thereof. Both the probe and targetnucleic acid can be present as a single strand, duplex, triplex, etc.Moreover, the nucleic acid can be modified at the base moiety, sugarmoiety, or phosphate backbone with other groups such as radioactivelabels, minor groove binders, intercalating agents, donor and/oracceptor moieties and the like.

For example, the nucleic acid can comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,nitroindole, and 2,6-diaminopurine.

In another embodiment, the nucleic acid comprises at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the nucleic acid comprises at least onemodified phosphate backbone selected from the group including, but notlimited to, a peptide nucleic acid hybrid, a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

Phosphodiester linked nucleic acids of the invention can be synthesizedby standard methods known in the art, e.g. by use of an automated DNAsynthesizer (such as are commercially available from P.E. Biosystems,etc.) using commercially available amidite chemistries. Nucleic acidsbearing modified phosphodiester linking groups can be synthesized bymethods known in the art. For example, phosphorothioate nucleic acidsmay be synthesized by the method of Stein et al. (Nucl. Acids Res.16:3209 (1988)), methylphosphonate nucleic acids can be prepared by useof controlled pore glass polymer supports (Sarin et al., Proc. Natl.Acad. Sci. U.S.A. 85:7448-7451 (1988)). Other methods of synthesizingboth phosphodiester- and modified phosphodiester-linked nucleic acidswill be apparent to those of skill in the art.

Nucleic acid probes of the invention can be synthesized by a number ofapproaches, e.g., Ozaki et al., Nucleic Acids Research, 20: 5205-5214(1992); Agrawal et al., Nucleic Acids Research, 18: 5419-5423 (1990); orthe like. The nucleic acid probes of the invention are convenientlysynthesized on an automated DNA synthesizer, e.g., a P.E. Biosystems,Inc. (Foster City, Calif.) model 392 or 394 DNA/RNA Synthesizer, usingstandard chemistries, such as phosphoramidite chemistry (see, forexample, disclosed in the following references, Beaucage et al.,Tetrahedron, 48: 2223-2311 (1992); Molko et al., U.S. Pat. No.4,980,460; Koster et al., U.S. Pat. No. 4,725,677; Caruthers et al.,U.S. Pat. Nos. 4,415,732; 4,458,066; and 4,973,679. Alternativechemistries resulting in non-natural backbone groups, such asphosphorothioate, phosphoramidate, and the like, can also be employed.

When the nucleic acids are synthesized utilizing an automated nucleicacid synthesizer, the donor and acceptor moieties are preferablyintroduced during automated synthesis. Alternatively, one or more ofthese moieties can be introduced either before or after the automatedsynthesis procedure has commenced. For example, donor and/or acceptorgroups can be introduced at the 3′-terminus using a solid supportmodified with the desired group(s). Additionally, donor and/or acceptorgroups can be introduced at the 5′-terminus by, for example a derivativeof the group that includes a phosphoramidite. In another exemplaryembodiment, one or more of the donor and/or acceptor groups isintroduced after the automated synthesis is complete.

In the dual labeled probes, the donor moiety is preferably separatedfrom the BHQ by at least about 10 nucleotides, and more preferably by atleast about 15 nucleotides. The donor moiety is preferably attached toeither the 3′- or 5′-terminal nucleotides of the probe. The BHQ moietyis also preferably attached to either the 3′- or 5′-terminal nucleotidesof the probe. More preferably, the donor and acceptor moieties areattached to the 3′- and 5′- or 5′- and 3′-terminal nucleotides of theprobe, respectively, although internal placement is also useful.

Once the desired nucleic acid is synthesized, it is preferably cleavedfrom the solid support on which it was synthesized and treated, bymethods known in the art, to remove any protecting groups present (e.g.,60° C., 5 h, concentrated ammonia). In those embodiments in which abase-sensitive group is attached to the nucleic acids (e.g., TAMRA), thedeprotection will preferably use milder conditions (e.g.,butylamine:water 1:3, 8 hours, 70° C.). Deprotection under theseconditions is facilitated by the use of quick deprotect amidites (e.g.,dC-acetyl, dG-dmf).

Following cleavage from the support and deprotection, the nucleic acidis purified by any method known in the art, including chromatography,extraction and gel purification. In a preferred embodiment, the nucleicacid is purified using HPLC. The concentration and purity of theisolated nucleic acid is preferably determined by measuring the opticaldensity at 260 mn in a spectrophotometer.

Peptide Probes

Peptides, proteins and peptide nucleic acids that are labeled with afluorophore and a quencher of the invention can be used in both in vivoand in vitro enzymatic assays.

Thus, in another aspect, the present invention provides a method fordetermining whether a sample contains an enzyme. The method comprises:(a) contacting the sample with a peptide construct; (b) exciting thefluorophore; and (c) determining a fluorescence property of the sample,wherein the presence of the enzyme in the sample results in a change inthe fluorescence property.

Peptide constructs useful in practicing the invention include those withthe following features: i) a fluorophore; ii) a BHQ of the invention;and iii) a cleavage or assembly recognition site for the enzyme.Moreover, the peptide construct is preferably of a length andorientation and in a conformation allowing donor-acceptor energytransfer between the fluorophore and the BHQ when the fluorophore isexcited.

When the probe is used to detect an enzyme, such as a degradative enzyme(e.g., protease), and a degree of donor-acceptor energy transfer that islower than an expected amount is observed, this is generally indicativeof the presence of an enzyme. The degree of donor-acceptor energytransfer in the sample can be determined, for example, as a function ofthe amount of fluorescence from the donor moiety, the amount offluorescence from the acceptor moiety, the ratio of the amount offluorescence from the donor moiety to the amount of fluorescence fromthe acceptor moiety or the excitation state lifetime of the donormoiety.

The assay also is useful for determining the amount of enzyme in asample by determining the degree of donor-acceptor energy transfer at afirst and second time after contact between the enzyme and the tandemconstruct, and determining the difference in the degree ofdonor-acceptor energy transfer. The difference in the degree ofdonor-acceptor energy transfer reflects the amount of enzyme in thesample.

The assay methods also can also be used to determine whether a compoundalters the activity of an enzyme, i.e., screening assays. Thus, in afurther aspect, the invention provides methods of determining the amountof activity of an enzyme in a sample from an organism. The methodincludes: (a) contacting a sample comprising the enzyme and the compoundwith a peptide construct comprising (b) exciting the fluorophore; and(c) determining a fluorescence property of the sample, wherein theactivity of the enzyme in the sample results in a change in thefluorescence property. Peptide constructs useful in this aspect of theinvention are substantially similar to those described immediatelyabove.

In a preferred embodiment, the amount of enzyme activity in the sampleis determined as a function of the degree of donor-acceptor energytransfer in the sample and the amount of activity in the sample iscompared with a standard activity for the same amount of the enzyme. Adifference between the amount of enzyme activity in the sample and thestandard activity indicates that the compound alters the activity of theenzyme.

Representative enzymes with which the present invention can be practicedinclude, for example, trypsin, enterokinase, HIV-1 protease, prohormoneconvertase, interleukin-1b-converting enzyme, adenovirus endopeptidase,cytomegalovirus assemblin, leishmanolysin, β-secretase for amyloidprecursor protein, thrombin, renin, angiotensin-converting enzyme,cathepsin-D and a kininogenase, and proteases in general.

Proteases play essential roles in many disease processes such asAlzheimer's, hypertension, inflammation, apoptosis, and AIDS. Compoundsthat block or enhance their activity have potential as therapeuticagents. Because the normal substrates of peptidases are linear peptidesand because established procedures exist for making non-peptidicanalogs, compounds that affect the activity of proteases are naturalsubjects of combinatorial chemistry. Screening compounds produced bycombinatorial chemistry requires convenient enzymatic assays.

The most convenient assays for proteases are based on donor-acceptorenergy transfer from a donor fluorophore to a quencher placed atopposite ends of a short peptide chain containing the potential cleavagesite (see, Knight C. G., Methods in Enzymol. 248:18-34 (1995)).Proteolysis separates the fluorophore and quencher, resulting inincreased intensity in the emission of the donor fluorophore. Existingprotease assays use short peptide substrates incorporating unnaturalchromophoric amino acids, assembled by solid phase peptide synthesis.

Assays of the invention are also useful for determining andcharacterizing substrate cleavage sequences of proteases or foridentifying proteases, such as orphan proteases. In one embodiment themethod involves the replacement of a defined linker moiety amino acidsequence with one that contains a randomized selection of amino acids. Alibrary of fluorescent BHQ-bearing probes, wherein the fluorophore andthe BHQ are linked by a randomized peptide linker moiety can begenerated using recombinant engineering techniques or syntheticchemistry techniques. Screening the members of the library can beaccomplished by measuring a signal related to cleavage, such asdonor-acceptor energy transfer, after contacting the cleavage enzymewith each of the library members of the tandem fluorescent peptideconstruct. A degree of donor-acceptor energy transfer that is lower thanan expected amount indicates the presence of a linker sequence that iscleaved by the enzyme. The degree of donor-acceptor energy transfer inthe sample can be determined, for example, as a function of the amountof fluorescence from the donor moiety, the amount of fluorescence fromthe acceptor donor moiety, or the ratio of the amount of fluorescencefrom the donor moiety to the amount of fluorescence from the acceptormoiety or the excitation state lifetime of the donor moiety.

In the tandem constructs of the invention, the donor and acceptormoieties are connected through a linker moiety. The linker moiety,preferably, includes a peptide moiety, but can be or can include anotherorganic molecular moiety, as well. In a preferred embodiment, the linkermoiety includes a cleavage recognition site specific for an enzyme orother cleavage agent of interest. A cleavage site in the linker moietyis useful because when a tandem construct is mixed with the cleavageagent, the linker is a substrate for cleavage by the cleavage agent.Rupture of the linker moiety results in separation of the fluorophoreand the quencher of the invention. The separation is measurable as achange in donor-acceptor energy transfer. Alternatively, peptideassembly can be detected by an increase in donor-acceptor energytransfer between a peptide fragment bearing a BHQ and a peptide fragmentbearing a donor moiety.

When the cleavage agent of interest is a protease, the linker generallyincludes a peptide containing a cleavage recognition sequence for theprotease. A cleavage recognition sequence for a protease is a specificamino acid sequence recognized by the protease during proteolyticcleavage. Many protease cleavage sites are known in the art, and theseand other cleavage sites can be included in the linker moiety. See,e.g., Matayoshi et al. Science 247: 954 (1990); Dunn et al. Meth.Enzymol. 241: 254 (1994); Seidah et al. Meth. Enzymol. 244: 175 (1994);Thornberry, Meth. Enzymol. 244: 615 (1994); Weber et al. Meth. Enzymol.244: 595 (1994); Smith et al. Meth. Enzymol. 244: 412 (1994); Bouvier etal. Meth. Enzymol. 248: 614 (1995), Hardy et al., in AMYLOID PROTEINPRECURSOR IN DEVELOPMENT, AGING, AND ALZHEIMER'S DISEASE, ed. Masters etal. pp. 190-198(1994).

Solid Support Immobilized BHQ Analogues

The BHQs of the invention can be immobilized on substantially anypolymer, biomolecule, and solid or semi-solid material having any usefulconfiguration. Moreover, any conjugate comprising one or more BHQs canbe similarly immobilized. When the support is a solid or semi-solid,examples of preferred types of supports for immobilization of thenucleic acid probe include, but are not limited to, controlled poreglass, glass plates, polystyrene, avidin coated polystyrene beads,cellulose, nylon, acrylamide gel and activated dextran. These solidsupports are preferred because of their chemical stability, ease offunctionalization and well-defined surface area. Solid supports such as,controlled pore glass (CPG, 500 Å, 1000 Å) and non-swelling highcross-linked polystyrene (1000 Å) are particularly preferred.

According to the present invention, the surface of a solid support isfunctionalized with a quencher of the invention or a species including aquencher of the invention. For clarity of illustration, the followingdiscussion focuses on attaching a reactive BHQ to a solid support. Thefollowing discussion is also broadly relevant to attaching a speciesthat includes within its structure a reactive BHQ to a solid support,and the attachment of such species and reactive BHQ analogues to othermolecules and structures.

The BHQs are preferably attached to a solid support by forming a bondbetween a reactive group on the BHQ and a reactive group on the surfaceof the solid support or a linker attached to the solid support, therebyderivatizing the solid support with one or more BHQ analogues. The bondbetween the solid support and the BHQ is preferably a covalent bond,although ionic, dative and other such bonds are useful as well. Reactivegroups which can be used in practicing the present invention arediscussed in detail above and include, for example, amines, hydroxylgroups, carboxylic acids, carboxylic acid derivatives, alkenes,sulfhydryls, siloxanes, etc.

A large number of solid supports appropriate for practicing the presentinvention are available commercially and include, for example, peptidesynthesis resins, both with and without attached amino acids and/orpeptides (e.g., alkoxybenzyl alcohol resin, aminomethyl resin,aminopolystyrene resin, benzhydrylamine resin, etc. (Bachem)),functionalized controlled pore glass (BioSearch Technologies, Inc.), ionexchange media (Aldrich), functionalized membranes (e.g., —COOHmembranes; Asahi Chemical Co., Asahi Glass Co., and Tokuyama Soda Co.),and the like.

Moreover, for applications in which an appropriate solid support is notcommercially available, a wide variety of reaction types are availablefor the functionalization of a solid support surface. For example,supports constructed of a plastic such as polypropylene, can be surfacederivatized by chromic acid oxidation, and subsequently converted tohydroxylated or aminomethylated surfaces. The functionalized support isthen reacted with a BHQ of complementary reactivity, such as a BHQactive ester, acid chloride or sulfonate ester, for example. Supportsmade from highly crosslinked divinylbenzene can be surface derivatizedby chloromethylation and subsequent functional group manipulation.Additionally, functionalized substrates can be made from etched, reducedpolytetrafluoroethylene.

When the support is constructed of a siliceous material such as glass,the surface can be derivatized by reacting the surface Si—OH, SiO—H,and/or Si—Si groups with a functionalizing reagent.

In a preferred embodiment, wherein the substrates are made from glass,the covalent bonding of the reactive group to the glass surface isachieved by conversion of groups on the substrate's surface by asilicon-modifying reagent such as:(R^(a)O)₃—Si—R^(b)—X^(a)  (VII)where R^(a) is an alkyl group, such as methyl or ethyl, R^(b) is alinking group between silicon and X^(a), and X^(a) is a reactive groupor a protected reactive group. Silane derivatives having halogens orother leaving groups beside the displayed alkoxy groups are also usefulin the present invention.

In another preferred embodiment, the reagent used to functionalize thesolid support provides for more than one reactive group per each reagentmolecule. Using reagents, such as the compound below, each reactive siteon the substrate surface is, in essence, “amplified” to two or morefunctional groups:(R^(a)O)₃—Si—R^(b)—(X^(a))_(n)  (VIII)where R^(a) is an alkyl group (e.g.,methyl, ethyl), R^(b) is a linkinggroup between silicon and X^(a), X^(a) is a reactive group or aprotected reactive group and n is an integer between 2 and 50, and morepreferably between 2 and 20. The amplification of a BHQ by itsattachment to a silicon-containing substrate is intended to be exemplaryof the general concept of BHQ amplification. This amplification strategyis equally applicable to other aspects of the invention in which a BHQanalogue is attached to another molecule or solid support.

A number of siloxane functionalizing reagents can be used, for example:

-   -   1. Hydroxyalkyl siloxanes (Silylate surface, functionalize with        diborane, and H₂O₂ to oxidize to the alcohol)        -   a. allyl trichlorosilane→→3-hydroxypropyl        -   b. 7-oct-1-enyl trichlorchlorosilane→→8-hydroxyoctyl    -   2. Diol(dihydroxyalkyl) siloxanes (silylate surface and        hydrolyze to diol)a. (glycidyl        trimethoxysilane→→(2,3-dihydroxypropyloxy)propyl    -   3. Aminoalkyl siloxanes (amines requiring no intermediate        functionalizing step)        -   a. 3-aminopropyl trimethoxysilane→aminopropyl    -   4. Dimeric secondary aminoalkyl siloxanes        -   a.            bis(3-trimethoxysilylpropyl)amine→bis(silyloxylpropyl)amine.

It will be apparent to those of skill in the art that an array ofsimilarly useful functionalizing chemistries is available when supportcomponents other than siloxanes are used. Thus, for example alkylthiols, functionalized as discussed above in the context ofsiloxane-modifying reagents, can be attached to metal films andsubsequently reacted with a BHQ to produce the immobilized compound ofthe invention.

R groups of use for R^(b) in the above described embodiments of thepresent invention include, but are not limited to, alkyl, substitutedalkyl, aryl, arylalkyl, substituted aryl, substituted arylalkyl, acyl,halogen, hydroxy, amino, alkylamino, acylamino, alkoxy, acyloxy,aryloxy, aryloxyalkyl, mercapto, saturated cyclic hydrocarbon,unsaturated cyclic hydrocarbon, heteroaryl, heteroarylalkyl, substitutedheteroaryl, substituted heteroarylalkyl, heterocyclic, substitutedheterocyclic and heterocyclicalkyl groups and combinations thereof.

Nucleic Acid Capture Probes

In one embodiment, an immobilized nucleic acid comprising a BHQ is usedas a capture probe. The nucleic acid probe can be attached directly to asolid support, for example by attachment of the 3′- or 5′-terminalnucleotide of the probe to the solid support. More preferably, however,the probe is attached to the solid support by a linker (i.e., spacerarm, supra). The linker serves to distance the probe from the solidsupport. The linker is most preferably from about 5 to about 30 atoms inlength, more preferably from about 10 to about 50 atoms in length.

In yet another preferred embodiment, the solid support is also used asthe synthesis support in preparing the probe. The length and chemicalstability of the linker between the solid support and the first 3′-unitof nucleic acid play an important role in efficient synthesis andhybridization of support bound nucleic acids. The linker arm should besufficiently long so that a high yield (>97%) can be achieved duringautomated synthesis. The required length of the linker will depend onthe particular solid support used. For example, a six atom linker isgenerally sufficient to achieve a >97% yield during automated synthesisof nucleic acids when high cross-linked polystyrene is used as the solidsupport. The linker arm is preferably at least 20 atoms long in order toattain a high yield (>97%) during automated synthesis when CPG is usedas the solid support.

Hybridization of a probe immobilized on a solid support generallyrequires that the probe be separated from the solid support by at least30 atoms, more preferably at least 50 atoms. In order to achieve thisseparation, the linker generally includes a spacer positioned betweenthe linker and the 3′-terminus. For nucleic acid synthesis, the linkerarm is usually attached to the 3′-OH of the 3′-terminus by an esterlinkage which can be cleaved with basic reagents to free the nucleicacid from the solid support.

A wide variety of linkers are known in the art, which may be used toattach the nucleic acid probe to the solid support. The linker may beformed of any compound, which does not significantly interfere with thehybridization of the target sequence to the probe attached to the solidsupport. The linker may be formed of, for example, a homopolymericnucleic acid, which can be readily added on to the linker by automatedsynthesis. Alternatively, polymers such as functionalized polyethyleneglycol can be used as the linker. Such polymers are presently preferredover homopolymeric nucleic acids because they do not significantlyinterfere with the hybridization of probe to the target nucleic acid.Polyethylene glycol is particularly preferred because it is commerciallyavailable, soluble in both organic and aqueous media, easy tofunctionalize, and completely stable under nucleic acid synthesis andpost-synthesis conditions.

The linkages between the solid support, the linker and the probe arepreferably not cleaved during synthesis or removal of base protectinggroups under basic conditions at high temperature. These linkages can,however, be selected from groups that are cleavable under a variety ofconditions. Examples of presently preferred linkages include carbamate,ester and amide linkages.

Acrylamide-Immobilized Probes

In another preferred embodiment, a species is within a matrix, such asan acrylamide matrix and the species bears a BHQ, or the presence of theimmobilized species is ascertained using a probe bearing a BHQ. In apreferred embodiment, the immobilization is accomplished in conjunctionwith the “acrydite” process invented and commercialized by MosaicTechnologies (Cambridge, Mass., see, Rehman et al., Nucleic AcidsResearch, 27: 649-655 (1999)). The acrydite method allows immobilizationof alkene labeled capture probes within a polymerized polyacrylamidenetwork. When target mixes are run past the immobilized probe band underelectrophoresis conditions, the target nucleic acid is capturedsubstantially quantitatively. However, detection of this event currentlyrequires a second probe. In one embodiment, probes bearing a BHQ, and/ora fluorophore, are immobilized in an acrylamide matrix and subsequentlycontacted with the target mix. By using fluorescent probes as captureprobes, signals from target mixes can be directly detected in real time.

Microarrays

The present invention also provides microarrays including immobilizedBHQs and compounds (e.g., peptides, nucleic acids, bioactive agents,etc.) functionalized with BHQs. Moreover, the invention provides methodsof interrogating microarrays using probes that are functionalized withBHQs. The immobilized species and the probes are selected fromsubstantially any type of molecule, including, but not limited to, smallmolecules, peptides, enzymes nucleic acids and the like.

Nucleic acid microarrays consisting of a multitude of immobilizednucleic acids are revolutionary tools for the generation of genomicinformation, see, Debouck et al., in supplement to Nature Genetics,21:48-50 (1999). The discussion that follows focuses on the use of BHQsin conjunction with nucleic acid microarrays. This focus is intended tobe illustrative and does not limit the scope of materials with whichthis aspect of the present invention can be practiced.

In another preferred embodiment, the compounds of the present inventionare utilized in a microarray format. The BHQs, or species bearing BHQscan themselves be components of a microarray or, alternatively they canbe utilized as a tool to screen components of a microarray.

Thus, in a preferred embodiment, the present invention provides a methodof screening a microarray. The method includes contacting the members ofthe microarray with, for example, a BHQ-bearing probe and interrogatingthe microarray for regions of fluorescence. In an exemplary embodiment,fluorescent regions are indicative of the presence of an interactionbetween the BHQ-bearing probe and a microarray component. In anotherversion of this method, the microarray is interrogated for regions inwhich fluorescence is quenched by the BHQ, again indicating the presenceof an interaction between the BHQ-bearing probe and a component of themicroarray.

In another preferred embodiment, the array comprises immobilizedBHQ-bearing donor-acceptor energy transfer probes as the interrogatingspecies. In this embodiment, the probe “turns on” when hybridized to itstarget. Such arrays are easily prepared and read, and can be designed togive quantitative data. Arrays comprising BHQ-bearing probes arevaluable tools for expression analysis and clinical genomic screening.

In another preferred embodiment, the immobilized BHQ-bearing probe isnot a donor-acceptor energy transfer probe. A microarray based on suchas format can be used to probe for the presence of interactions betweenan analyte and the immobilized probe by, for example, observing thequenching of analyte fluorescence upon interaction between the probe andanalyte.

In a further preferred embodiment, the microarrays comprise n regionsthat comprise identical or different species (e.g., nucleic acidsequences, bioactive agents). For example, the microarray can comprise amixture of n regions comprising groups of identical species. In apreferred embodiment, n is a number from 2 to 100, more preferably, from10 to 1,000, and more preferably from 100 to 10,000. In a still furtherpreferred embodiment, the n regions are patterned on a substrate as ndistinct locations in a manner that allows the identity of each of the nlocations to be ascertained.

In yet another preferred embodiment, the invention also provides amethod for preparing a microarray of n BHQ-bearing probes. The methodincludes attaching BHQ-bearing probes to selected regions of asubstrate. A variety of methods are currently available for makingarrays of biological macromolecules, such as arrays nucleic acidmolecules. The following discussion focuses on the assembly of amicroarray of BHQ-bearing probes, this focus is for reasons of brevityand is intended to be illustrative and not limiting.

One method for making ordered arrays of BHQ-bearing probes on asubstrate is a “dot blot” approach. In this method, a vacuum manifoldtransfers a plurality, e.g., 96, aqueous samples of probes from 3millimeter diameter wells to a substrate. The probe is immobilized onthe porous membrane by baking the membrane or exposing it to UVradiation. A common variant of this procedure is a “slot-blot” method inwhich the wells have highly-elongated oval shapes.

Another technique employed for making ordered arrays of probes uses anarray of pins dipped into the wells, e.g., the 96 wells of a microtiterplate, for transferring an array of samples to a substrate, such as aporous membrane. One array includes pins that are designed to spot amembrane in a staggered fashion, for creating an array of 9216 spots ina 22×22 cm area. See, Lehrach, et al., HYBRIDIZATION FINGERPRINTING INGENOME MAPPING AND SEQUENCING, GENOME ANALYSIS, Vol. 1, Davies et al,Eds., Cold Springs Harbor Press, pp. 39-81 (1990).

An alternate method of creating ordered arrays of probes is analogous tothat described by Pirrung et al. (U.S. Pat. No. 5,143,854, issued 1992),and also by Fodor et al., (Science, 251: 767-773 (1991)). This methodinvolves synthesizing different probes at different discrete regions ofa particle or other substrate. This method is preferably used withrelatively short probe molecules, e.g., less than 20 bases. A relatedmethod has been described by Southern et al. (Genomics, 13: 1008-1017(1992)).

Khrapko, et al., DNA Sequence, 1: 375-388 (1991) describes a method ofmaking an nucleic acid matrix by spotting DNA onto a thin layer ofpolyacrylamide. The spotting is done manually with a micropipette.

The substrate can also be patterned using techniques such asphotolithography (Kleinfield et al., J. Neurosci. 8:4098-120 (1998)),photoetching, chemical etching and microcontact printing (Kumar el al.,Langmuir 10:1498-511 (1994)). Other techniques for forming patterns on asubstrate will be readily apparent to those of skill in the art.

The size and complexity of the pattern on the substrate is limited onlyby the resolution of the technique utilized and the purpose for whichthe pattern is intended. For example, using microcontact printing,features as small as 200 nm are layered onto a substrate. See, Xia, Y.,J. Am. Chem. Soc. 117:3274-75 (1995). Similarly, using photolithography,patterns with features as small as 1 μm are produced. See, Hickman etal., J. Vac. Sci. Technol. 12:607-16 (1994). Patterns which are usefulin the present invention include those which include features such aswells, enclosures, partitions, recesses, inlets, outlets, channels,troughs, diffraction gratings and the like.

In a presently preferred embodiment, the patterning is used to produce asubstrate having a plurality of adjacent wells, indentations or holes tocontain the probes. In general, each of these substrate features isisolated from the other wells by a raised wall or partition and thewells do not readily fluidically communicate. Thus, a particle, reagentor other substance, placed in a particular well remains substantiallyconfined to that well. In another preferred embodiment, the patterningallows the creation of channels through the device whereby an analyte orother substance can enter and/or exit the device.

In another embodiment, the probes are immobilized by “printing” themdirectly onto a substrate or, alternatively, a “lift off” technique canbe utilized. In the lift off technique, a patterned resist is laid ontothe substrate, and a probe is laid down in those areas not covered bythe resist and the resist is subsequently removed. Resists appropriatefor use with the substrates of the present invention are known to thoseof skill in the art. See, for example, Kleinfield et al., J. Neurosci.8:4098-120 (1998). Following removal of the photoresist, a second probe,having a structure different from the first probe can be bonded to thesubstrate on those areas initially covered by the resist. Using thistechnique, substrates with patterns of probes having differentcharacteristics can be produced. Similar substrate configurations areaccessible through microprinting a layer with the desiredcharacteristics directly onto the substrate. See, Mrkish et al. Ann.Rev. Biophys. Biomol. Struct. 25:55-78 (1996).

Spacer Groups

As used herein, the term “spacer group,” refers to constituents ofBHQ-bearing probes. The spacer group links donor and/or acceptormoieties and other groups to the nucleic acid, peptide or othercomponent of the probe. The spacer groups can be hydrophilic (e.g.,tetraethylene glycol, hexaethylene glycol, polyethylene glycol) or theycan be hydrophobic (e.g., hexane, decane, etc.).

In a preferred embodiment, using solid supports the immobilizedconstruct includes a spacer between the solid support reactive group andthe BHQ analogue. The linker is preferably selected from C₆-C₃₀ alkylgroups, C₆-C₃₀ substituted alkyl groups, polyols, polyethers (e.g.,poly(ethyleneglycol)), polyamines, polyamino acids, polysaccharides andcombinations thereof.

In certain embodiments, it is advantageous to have the donor and/oracceptor of the probe attached to another polymeric component by a groupthat provides flexibility and distance from the polymeric component.Using such spacer groups, the properties of the donor and/or acceptoradjacent to another probe component is modulated. Properties that areusefully controlled include, for example, hydrophobicity,hydrophilicity, surface-activity, the distance of the donor and/or BHQmoiety from the other probe components (e.g., carrier molecule) and thedistance of the donor from the BHQ.

In an exemplary embodiment, the spacer serves to distance the BHQ from anucleic acid. Spacers with this characteristic have several uses. Forexample, a BHQ held too closely to the nucleic acid may not interactwith the donor group, or it may interact with too low of an affinity.When a BHQ is itself sterically demanding, the interaction leading toquenching can be undesirably weakened, or it may not occur at all, dueto a sterically-induced hindering of the approach of the two components.

When the construct comprising the BHQ is immobilized by attachment to,for example, a solid support, the construct can also include a spacermoiety between the reactive group of the solid support and the BHQanalogue, or other probe component bound to the solid support.

In yet a further embodiment, a spacer group used in the probes of theinvention is provided with a group that can be cleaved to release abound moiety, such as, for example, a BHQ, fluorophore, minor groovebinder, intercalating moiety, and the like from the polymeric component.Many cleaveable groups are known in the art. See, for example, Jung etal., Biochem. Biophys. Acta, 761: 152-162 (1983); Joshi et al., J. Biol.Chem., 265: 14518-14525 (1990); Zarling et al., J. Immunol., 124:913-920 (1980); Bouizar et al., Eur. J. Biochem., 155: 141-147 (1986);Park et al., J. Biol. Chem., 261: 205-210 (1986); Browning et al., J.Immunol., 143: 1859-1867 (1989). Moreover a broad range of cleavable,bifunctional (both homo- and hetero-bifunctional) spacer arms arecommercially available from suppliers such as Pierce.

An exemplary embodiment utilizing spacer groups is set forth in FormulaeVII and VIII, above. In these formulae, R^(b) is either stable or it canbe cleaved by chemical or photochemical reactions. For example, R^(b)groups comprising ester or disulfide bonds can be cleaved by hydrolysisand reduction, respectively. Also within the scope of the presentinvention is the use of R^(b) groups which are cleaved by light such as,for example, nitrobenzyl derivatives, phenacyl groups, benzoin esters,etc. Other such cleaveable groups are well-known to those of skill inthe art.

Kits

In another aspect, the present invention provides kits containing one ormore of the BHQs or BHQ-bearing compositions of the invention. In oneembodiment, a kit will include a reactive BHQ derivative and directionsfor attaching this derivative to another molecule. In anotherembodiment, the kit includes a BHQ-labeled nucleic acid that optionallyis also labeled with a fluorophore and directions for using this nucleicacid in one or more assay formats. Other formats for kits will beapparent to those of skill in the art and are within the scope of thepresent invention.

The materials and methods of the present invention are furtherillustrated by the examples which follow. These examples are offered toillustrate, but not to limit the claimed invention.

EXAMPLES

The following Examples illustrate the synthesis and characterization ofexemplary species of the invention.

Example 1 sets for the synthesis of the quencher BH1 and its conversioninto a controlled pore glass conjugate. Examples 2 and 3, providesimilar details regarding the quenchers BH2 and BH3.

Example 4 sets forth the incorporation of exemplary quenchers of theinvention into nucleic acid-based donor-acceptor energy transfer probes.The quenching efficiency of the BHQs in the probes is assessed andcompared to that of the art-recognized dark quencher DABCYL.

Example 1

This Example sets forth the synthesis and characterization of BH1 andderivatives thereof.

1.1 Synthesis of4-methyl-2-nitrobenzylazo-2′-methyl-5′-nitrobenzylazo-4″-N,N-di(2-hydroxyethyl)azobenzene,(BH1 diol), 6

To a rapidly stirred suspension of 25 g (60 mmol) Fast Corinth V salt(Aldrich 22,736-5) in 400 mL of chilled water (ice bath) was added 50 g(276 mmol) N-phenyldiethanolamine dissolved in 400 mL methanol and 300mL sat'd NaHCO₃ over 20 min. The mixture changed color from yellow todeep red during the course of the addition. The mixture was chilled foran additional 1 hr after addition, then filtered through a medium glassfrit. The dark red solid was washed with 300 ml of ice cold water andair dried for 3 days. The yield of 6 was 27 g, (91%). TLC rf 0.15(Silica plate, 5% MeOH in CH₂Cl₂). MALDI M/e 493.11 (Calc'd 492.5). ¹HNMR (CDCl₃, δ) 7.9 (d, 2H), 7.65 (m, 2H), 7.6 (s, 1H), 7.45(d, 1H),7.4(s, 1H), 6.8(d, 2H), 4.0(s, 3H), 3.95(t, 2H), 3.85(t, 2H), 3.7(t,2H), 3.6(t, 2H), 2.7(s, 3H), 2.5(s, 3H).

1.2 Synthesis of4-methyl-2-nitrobenzylazo-2′-methyl-5′-nitrobenzylazo-4″-N,N-(2-hydroxyethyl)-(2-O-(4,4′dimethoxytrityl)ethylazobenzene(DMT-BH1), 7

A solution of 25 g (50 mmol) 6 in 400 mL of dry pyridine was stripped todryness, and 7 g (21 mmol) of DMT-chloride was added in 300 mL of drypyridine. The solution sat for 3 days at rt, then was stripped to a tar.The black residue was dissolved in 600 mL ethyl acetate, and washed with300 mL of 1 N aqueous citric acid, followed by a 300 mL sat'd aqueousNaHCO₃ wash. The organic layer was dried over MgSO₄ and stripped to atar. The residue was loaded onto a 55 by 8 cm chromatography column ofneutral alumina, 7% by weight water, and eluted first with a mobilephase of 0.5% MeOH, 0.5% pyridine in CH₂Cl₂. After 1 L of solventeluted, a gradient to 2% MeOH was run over 4 L. Fractions containingpure 7 (0.42 rf, silica plate, 2% MeOH, 2% pyridine in CH₂Cl₂) werepooled and evaporated. The yield was 2.8 g (17% yield) of 2 as a darkfoam. MALDI M/e 793.88 (Calc'd 794.5). ¹H NMR (CDCl₃, δ) 7.85 (d, 2H),7.7 (m, 4H), 7.6 (s, 1H), 7.45(d, 1H), 7.4-7.1 (m, 10H), 6.8(m, 6H),4.0(s, 3H), 3.85(m, 2H), 3.75 (s, 6H), 3.7(m, 2H), 3.5-3.3(m, 4H),2.7(s, 3H), 2.5(s, 3H).

1.3 Synthesis of4-methyl-2-nitrobenzylazo-2′-methyl-5′-nitrobenzylazo-4″-N,N-(2-O-(N′,N′-diisopropyl-2-cyanoethylphosphite)ethyl)-(2-O-4,4′dimethoxytrityl)-ethyl)azobenzene,(DMT-BH1 amidite), 8

A solution of 2.8 g (3.5 mmol) of 7 in 50 mL of dry pyridine wasevaporated to dryness and high vacuum applied for several hrs. Asolution of 1.5 g (5 mmol) ofN,N,N′,N′-tetraisopropyl-2-cyanoethylphosphane and 60 mg of tetrazolewere mixed in 20 mL of dry acetonitrile and added to the flaskcontaining the dried 7. After 2 hrs, the solvent was stripped, and theresidue was dissolved in 200 mL of EtOAc. The organic layer was washedwith 100 mL sat'd aqueous NaHCO₃ and dried over MgSO₄. The solvent wasevaporated, and the residue applied to a 25 by 3 cm chromatographycolumn of neutral alumina, 7% by weight water, and eluted with a mobilephase of 75% Pet. ether, 23% EtOAc, 2% pyridine. Fractions containingpure 8 (0.67 rf, pre-run silica plate, 50% Pet. ether, 48% EtOAc, 2%pyridine) were pooled and evaporated. The yield was 1 g (20% yield) of 8as a dark foam. MALDI M/e 995.5 (Calc'd 994.5).

1.4 Synthesis of BH1-CPG, 9

11 g DMT-2,2′-sulfonyldiethanol-succinyl controlled pore glass, 500Å,Biosearch part # BGS-5000, was washed three times with 3% dichloroaceticacid to effect removal of the DMT group. The CPG was washed well withthree 50 mL portions of CH₂Cl₂, followed by one 50 mL wash with drypyridine. The CPG was added to the flask containing 1 g of 8.25 mL ofdry pyridine was added, and the solvent was removed by rotaryevaporation, followed by high vacuum for 3 hrs. The dried CPG andamidite were treated with a solution of 1 g S-ethyl tetrazole in 20 mLof dry acetonitrile for 20 min. The CPG was washed in a sintered glassfunnel twice with 50 mL of acetonitrile, then 50 mL of 0.02 M iodine in90% THF, 8% water and 2% pyridine were added. After 5 min, the iodinesolution was washed out of the CPG with three 50 mL portions ofacetonitrile, then a capping solution of 10% Ac₂O, 10% N-methylimidazoleand 10% pyridine in THF was added. After 20 min, the solution was washedout of the CPG with three 50 mL portions of acetonitrile, followed bythree 50 mL portions of CH₂Cl₂. The material was dried overnight underhigh vacuum. DMT loading determination on the dried material was 16μM/g.

Example 2

This Example sets forth the synthesis and characterization of BH2 andderivatives thereof.

2.1 Synthesis of 4-nitrobenzylazo-2′,5′-dimethoxybenzylazo-4″-N,N-di(2-hydroxyethyl) azobenzene, (BH2 diol), 10

To a rapidly stirred suspension of 25 g (60 mmol) Fast Black K salt(Aldrich 20,151-0) in 400 mL of chilled water (ice bath) was added 50 g(276 mmol) N-phenyldiethanolamine dissolved in 400 mL methanol and 300mL sat'd NaHCO₃ over 20 min. The mixture changed color from brown todeep blue during the course of the addition. The mixture was chilled foran additional 1 hr after addition, then filtered through a medium glassfrit. The dark blue solid was washed with 300 ml of ice cold water andair dried for 3 days. The yield of 10 was 22 g, (74%). TLC rf 0.2(Silica plate, 5% MeOH in CH₂Cl₂). MALDI M/e 493.13 (Calc'd 494.4). ¹HNMR (CDCl₃, δ) 8.3 (d, 2H), 8.0(d, 2H), 7.85(d, 2H), 7.4(d, 2H), 6.7(m,2H), 4.1(s, 3H), 3.95(s, 3H), 3.9(t, 2H), 3.8(t, 2H), 3.7(t, 2H), 3.5(t,3H).

2.2 Synthesis of4-nitrobenzylazo-2′,5′-dimethoxybenzylazo-4″-N,N-(2-hydroxy-ethyl)-(2-O-(4,4′dimethoxytrityl)ethylazobenzene1,(DMT-BH2), 11

A solution of 22 g (44 mmol) 10 in 400 mL of dry pyridine was strippedto dryness, and 7 g (21 mmol) of DMT-chloride was added in 300 mL of drypyridine. The solution sat for 3 days at rt, then was stripped to a tar.The black residue was dissolved in 600 mL ethyl acetate, and washed with300 mL of 1 N aqueous citric acid, followed by a 300 mL sat'd aqueousNaHCO₃ wash. The organic layer was dried over MgSO₄ and stripped to atar. The residue was loaded onto a 55 by 8 cm chromatography column ofneutral alumina, 7% by weight water, and eluted first with a mobilephase of 0.5% MeOH, 0.5% pyridine in CH₂Cl₂. After 1 L of solventeluted, a gradient to 2% MeOH was run over 4 L. Fractions containingpure 11 (0.4 rf, silica plate, 2% MeOH, 2% pyridine in CH₂Cl₂) werepooled and evaporated. The yield was 2.8 g (17% yield) of 11 as a darkfoam. MALDI M/e 794.2 (Calc'd 796.4). ¹H NMR (CDCl₃, δ) 8.3 (d, 2H),7.9(d, 2H), 7.75(d, 2H), 7.4-7.1(m, 9H), 6.7-6.5(m, 6H), 4.1(s, 3H),3.95(s, 3H), 3.9-3.6(m, 12H), 3.3(t, 3H).

2.3 Synthesis of4-nitrobenzylazo-2′,5′-dimethoxybenzylazo-4″-N,N-(2-O-(N′,N′-diisopropyl-2-cyanoethylphosphite)ethyl)-(2-O-(4,4′dimethoxy-trityl)ethyl)-azobenzene,(DMT-BH2 amidite), 12

A solution of 2.8 g (3.5 mmol) 11 in 50 mL of dry pyridine wasevaporated to dryness and high vacuum applied for several hrs. Asolution of 1.5 g (5 mmol) ofN,N,N′,N′-tetraisopropyl-2-cyanoethylphosphane and 60 mg of tetrazolewere mixed in 20 mL of dry acetonitrile and added to the flaskcontaining the dried 11. After 2 hrs, the solvent was stripped, and theresidue was dissolved in 200 mL of EtOAc. The organic layer was washedwith 100 mL sat'd aqueous NaHCO₃ and dried over MgSO₄. The solvent wasevaporated, and the residue applied to a 25 by 3 cm chromatographycolumn of neutral alumina, 7% by weight water, and eluted with a mobilephase of 75% Pet. ether, 23% EtOAc, 2% pyridine. Fractions containingpure 12 (0.71 rf, pre-run silica plate, 50% Pet. ether, 48% EtOAc, 2%pyridine) were pooled and evaporated. The yield was 1 g (20% yield) of11 as a dark foam. MALDI M/e 996.06 (Calc'd 996.4).

2.4 Synthesis of BH2-CPG, 13

10 g DMT-2,2′-sulfonyldiethanol-succinyl controlled pore glass, 500Å,Biosearch part # BG5-5000, was washed three times with 3% dichloroaceticacid to effect removal of the DMT group. The CPG was washed well withthree 50 mL portions of CH₂Cl₂, followed by one 50 mL wash with drypyridine. The CPG was added to the flask containing 1 g of 12.25 mL ofdry pyridine was added, and the solvent was removed by rotaryevaporation, followed by high vacuum for 3 hrs. The dried CPG andamidite were treated with a solution of 1 g S-ethyl tetrazole in 20 mLof dry acetonitrile for 20 min. The CPG was washed in a sintered glassfunnel twice with 50 mL of acetonitrile, then 50 mL of 0.02 M iodine in90% THF, 8% water and 2% pyridine were added. After 5 min, the iodinesolution was washed out of the CPG with three 50 mL portions ofacetonitrile, then a capping solution of 10% Ac₂O, 10% N-methylimidazoleand 10% pyridine in THF was added. After 20 min, the solution was washedout of the CPG with three 50 mL portions of acetonitrile, followed bythree 50 mL portions of CH₂Cl₂. The material was dried overnight underhigh vacuum. DMT loading determination on the dried material was 30μM/g.

Example 3

This Example sets forth the synthesis and characterization of BH3 andderivatives thereof.

3.1 Synthesis of3-diethylamino-5-phenylphenazium-7-(4′-N,N-di(2-hydroxyethyl)azobenzene)chloride, (BH3 diol), 14

3-amino-7-(diethylamino)-5-phenylphenazium chloride (methylene violet3RAX, Aldrich 30,750-5), 10 g (26 mmol) was stirred in 200 mL 1 N HClwhile in an ice bath. A solution of 2 g NaNO₂ in 20 mL of cold water wasadded dropwise over 20 min. The solution was stirred for 30 min.N-phenyldiethanolamine, 4.7 g (27 mmol), was dissolved in 100 mL ofmethanol and added to the methylene violet solution, after the pH wasadjusted to 6 with NaOH solution. The solution changed color from violetto dark green. The solution was stirred for 1 hr, then extracted threetimes with 200 mL of CH₂Cl₂. The aqueous layer was evaporated, and thedark green solid was triturated with 3 200 mL portions of pyridine. Thepyridine was evaporated to give 2.9 g (21% yield) of 14 as a dark greensolid. (0.85 rf, silica plate, 15% MeOH, 2% pyridine in CH₂Cl₂) MALDIM/e 535.6 (calc'd 535.75).

3.2 Synthesis of3-diethylamino-5-phenylphenazium-7-(4′-N,N-(2-hydroxyethyl)-(2-O-(4,4′dimethoxytrityl)ethylazobenzenechloride, (DMT-BH3), 15

Compound 14, 2.9 g (5.4 mmol) was dried by stripping with 100 mL drypyridine, and then re-dissolved in 100 mL dry pyridine along with 2 g(5.9 mmol) of DMT chloride. The mixture sat overnight, and the solventwas stripped. The residue was re-dissolved in 200 mL CH₂Cl₂ and washedwith 200 mL 1 M aqueous citric acid. The aqueous layer was backwashedwith two 200 mL portions of CH₂Cl₂, and the combined organic layers wereevaporated to a tar. The residue was loaded onto a 20 by 3 cmchromatography column of neutral alumina, 7% by weight water, and elutedfirst with a mobile phase of 2% MeOH, 1% pyridine in CH₂Cl₂. A gradientto 6% MeOH was run over 3 L. Fractions containing pure 15 (0.9 rf,silica plate, 15% MeOH, 1% pyridine in CH₂Cl₂) were pooled andevaporated. The yield was 0.5 g (11% yield) of 15 as a dark foam. MALDIM/e 837.6 (calc'd 836.75)

3.3 Synthesis of3-diethylamino-5-phenylphenazium-7-(4′-N,N-(2-O-(N′,N′-diisopropyl-2-cyanoethylphosphite)ethyl)-(2-O-(4,4′dimethoxy-trityl)ethyl)azobenzenechloride, (DMT-BH3 amidite), 16

A solution of 0.5 g (0.6 mmol) 15 in 50 mL of dry pyridine wasevaporated to dryness and high vacuum applied for several hrs. Asolution of 0.5 g (1.7 mmol) ofN,N,N′,N′-tetraisopropyl-2-cyanoethylphosphane and 20 mg of tetrazolewere mixed in 20 mL of dry acetonitrile and added to the flaskcontaining the dried 15. After 2 hrs, the solvent was stripped, and theresidue was dissolved in 100 mL of EtOAc. The organic layer was washedwith 50 mL sat'd aqueous NaHCO₃ and dried over MgSO₄. The solvent wasevaporated, and the residue applied to a 15 by 3 cm chromatographycolumn of neutral alumina, 7% by weight water, and eluted first with amobile phase of 1% MeOH, 1% pyridine in CH₂Cl₂. A gradient to 6% MeOHwas run over 2 L. Fractions containing pure 16 (0.82 rf, pre-run silicaplate, 5% MeOH, 1% pyridine in CH₂Cl₂) were pooled and evaporated. Theyield was 0.26 g (42% yield) of 16 as a dark foam. MALDI M/e 1037.3(Calc'd 1036.75).

Synthesis of BH3-CPG, 17

2 g DMT-2,2′-sulfonyldiethanol-succinyl controlled pore glass, 500Å,Biosearch part # BG5-5000, was washed three times with 3% dichloroaceticacid to effect removal of the DMT group. The CPG was washed well withthree 20 mL portions of CH₂Cl₂, followed by one 20 mL wash with drypyridine. The CPG was added to the flask containing 0.26 g of 16.25 mLof dry pyridine was added, and the solvent was removed by rotaryevaporation, followed by high vacuum for 3 hrs. The dried CPG andamidite were treated with a solution of 0.5 g S-ethyl tetrazole in 10 mLof dry acetonitrile for 20 min. The CPG was washed in a sintered glassfunnel twice with 20 mL of acetonitrile, then 20 mL of 0.02 M iodine in90% THF, 8% water and 2% pyridine were added. After 5 min, the iodinesolution was washed out of the CPG with three 20 mL portions ofacetonitrile, then a capping solution of 10% Ac₂O, 10% N-methylimidazoleand 10% pyridine in THF was added. After 20 min, the solution was washedout of the CPG with three 20 mL portions of acetonitrile, followed bythree 20 mL portions of CH₂Cl₂. The material was dried overnight underhigh vacuum. DMT loading determination on the dried material was 12μM/g.

Example 4

This Example sets forth the preparation and characterization of nucleicacid analogues of exemplary BHQs of the invention. The nucleic acid-BHQconjugates are compared to a similar conjugate of DABCYL.

The efficiency of donor-acceptor energy transfer (FRET) is inverselyproportional to the sixth power of the distance between the donor andacceptor molecules (Stryer, L. Annu. Rev. Biochem. 1978, 47, 819-846).This property can be utilized to monitor hybridization of nucleic acidslabeled at one end with a donor fluorophore (reporter) and at theopposite end with an acceptor (quencher) (Parkhurst et al., Biochemistry1995, 34, 285-292). In the absence of a complementary target sequencethe dual labeled probe is very flexible and undergoes rapidconformational changes so that the average distance between the donor(D) and acceptor (A) is close enough for efficient FRET to occur. Uponhybridization to complementary target, a rigid DNA duplex is formedseparating the D-A pair and reducing the efficiency of transfer. Thismanifests itself in an increase in the fluorescence intensity of thereporter.

4.1 Synthesis and Characterization of BHQ Probes

To evaluate the efficiency of the Black Hole Quencher dyes (BHQs), theability of these dyes to quench a series of common fluorophores wascompared to the standard quencher dyes DABCYL and TAMRA in acomplementation assay. As TAMRA exhibits its own native fluorescence, itcan only be used to quench lower wavelength dyes and thus was only usedto quench FAM in this assay.

4.1a Materials and Methods

Nucleic acids were synthesized on a Biosearch 8700 automated DNAsynthesizer using standard phosphoramidite chemistry. A^(BZ), C^(AC), T,and G^(DMF) phosphoramidites were supplied by Chruachem. TAMRA, DABCYL,and BHQ controlled pore glass (CPG) supports from Biosearch Technologieswere used for attachment of quencher dyes to the 3′ terminus of nucleicacids. 5′ fluorophore labeling was accomplished using fluorophorephosphoramidites with the exception of Cy5 which was added as asuccinimidyl ester to 5′ amino nucleic acids using the manufacturers'protocol. 6-FAM and TFA-aminohexyl amidite were from Biosearch. Cy3phosphoramidite and Cy5 succinimidyl ester were from Amersham PharmaciaBiotech. Cleavage and deprotection of oligos was carried out in ammoniaat 60° C. for 3 hrs, with the exception of 3′ TAMRA oligos which weredeprotected in 1:3 t-butylamine:H₂O for 8 hours at 60° C., and 3′ BH3oligos which were deprotected for 1 hour at 60° C. in ammonia. Followingdeprotection, dual labeled probes were evaporated to dryness thenresuspended in HPLC grade water and filtered through 0.45 μM filters toprepare for HPLC purification. A two step method of HPLC purification ofanion exchange followed by reverse phase HPLC was used to purify alldual labeled probes. Purified probes were analyzed by both anionexchange and reverse phase HPLC. Complementary nucleic acid was purifiedon a Biosearch Micropure II reverse phase cartridge using the standardprotocol.

All fluorescence measurements were taken using a Spectramax Geminifluorescent microplate reader. Probes were dissolved at 200 nM in abuffer composed of of 10 mM Tris-HCl, 50 mM KCl, and 3.5 mM MgCl₂, pH8.3, both in the presence and absence of a five fold excess of perfectlycomplimentary nucleic acid. Fluorescein was excited at 470 nm andemission was read at 530 nm with a 515 nm cutoff filter in place. Cy3was excited at 510 nm and read at 567 nm with a 550 nm cutoff filter.Cy5 was excited at 630 nm and read at 660 nm with a 630 nm cutofffilter. Average fluorescent intensity from a set of eight buffer blankswas subtracted from all probe fluorescence measurements beforecalculation of signal to noise ratios.

The set of nucleic acids displayed in Table 2 was synthesized andrigorously purified by HPLC:

TABLE 2 Dual labeled probes prepared for hybridization assay. Reporterem max Quencher (5′) (nm) Sequence (5′ to 3′) (3′) FAM 518 ATG CCC TCCCCC ATG CCA TCC TAMRA TGC G (SEQ ID NO.:1) FAM 518 ATG CCC TCC CCC ATGCCA TCC DABCYL TGC G (SEQ ID NO.:1) FAM 518 ATG CCC TCC CCC ATG CCA TCCBH1 TGC G (SEQ ID NO.:1) FAM 518 ATG CCC TCC CCC ATG CCA TCC BH2 TGC G(SEQ ID NO.:1) Cy3 573 ATG CCC TCC CCC ATG CCA TCC DABCYL TGC G (SEQ IDNO.:1) Cy3 573 ATG CCC TCC CCC ATG CCA TCC BH1 TGC G (SEQ ID NO.:1) Cy3573 ATG CCC TCC CCC ATG CCA TCC BH2 TGC G (SEQ ID NO.:1) Cy5 678 ATG CCCTCC CCC ATG CCA TCC DABCYL TGC G (SEQ ID NO.:1) Cy5 678 ATG CCC TCC CCCATG CCA TCC BH2 TGC G (SEQ ID NO.:1) Cy5 678 ATG CCC TCC CCC ATG CCA TCCBH3 TGC G (SEQ ID NO.:1)

The signal to noise ratio (S:N) of hybridization for each probe wasmeasured in the presence of a five fold molar excess of perfectlycomplementary nucleic acid. All hybridization assays were performed in abuffer composed of 10 mM Tris-HCl, 50 mM KCl, and 3.5 mM MgCl₂, pH 8.3.S:N was calculated by dividing the fluorescence intensity of the probein the presence of complement by the fluorescence intensity of the probealone after subtracting out the fluorescence intensity of the bufferblank from each. All fluorescence measurements were taken in triplicateusing a Spectramax Gemini fluorescent plate reader.

4.2 Results

Improved quenching by the BHQs was demonstrated for three widelyseparated fluorophores. A two fold increase in signal to noise ratio isachieved by replacing DABCYL with BH1 for the fluorescein probe. S:Nincreases are more striking for the cyanine dye labeled probes, withapproximately a ten fold increase in S:N observed for the BH2 quenchedCy3 probe and a thirty fold S:N increase for the BH3 quenched Cy5 probeover identical structures quenched with DABCYL. This is consistant withthe theory that efficiency of FRET is proportional to the magnitude ofoverlap between the donor and acceptor (Haugland et al., Proc. Natl.Acad. Sci. U.S.A. 1969, 63, 23-30). DABCYL, which absorbs maximally at474 nm, quenches the red shifted reporter dyes with decreasingefficiency, whereas the BHQs can be chosen accordingly to have maximumspectral overlap with the reporter of interest.

Data from the hybridization assay is presented in Table 3.

TABLE 3 Signal to noise ratios of dual labeled FRET probes Noise Signalmeasurement no. Average Average donor/acceptor #1 #2 #3 #1 #2 #3 NoiseSignal S:N FAMTAM 89.18 91.50 89.54 279.13 277.08 272.78 90.08 276.333.07 FAMDAB 89.97 81.03 86.74 348.98 323.82 352.00 85.92 341.61 3.98FAMBH1 39.24 37.05 38.85 296.53 316.24 311.15 38.38 307.97 8.02 FAMBH267.12 66.64 56.79 445.48 415.78 442.75 63.52 434.34 6.84 Cy3DAB 19.6619.65 19.39 147.70 139.83 154.35 19.57 147.29 7.53 Cy3BH1 1.86 2.09 2.41140.72 135.20 135.25 2.12 137.06 64.51 Cy3BH2 2.02 1.94 2.08 149.65148.89 150.40 2.02 149.65 74.15 Cy5/DAB 57.09 59.15 50.50 157.36 176.04216.59 55.58 183.33 3.30 Cy5/BH2 19.92 21.47 22.53 263.64 288.20 256.0721.31 269.31 12.64 Cy5/BH3 1.65 2.17 1.55 197.96 210.71 208.06 1.79205.58 114.79

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto included within the spirit and purview of this application and areconsidered within the scope of the appended claims. All publications,patents, and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

1. A method for detecting a nucleic acid target sequence, said methodcomprising: (a) contacting said target sequence with a detector nucleicacid; comprising a single-stranded target binding sequence, saiddetector nucleic acid having linked thereto, i) a fluorophore; and ii) aquencher of excited state energy having a structure comprising at leastthree residues selected from aryl, substituted aryl, heteroaryl,substituted heteroaryl and combinations thereof, wherein at least two ofsaid residues are covalently linked via an exocyclic diazo bond, wherein said detector nucleic acid is in a conformation allowingdonor-acceptor energy transfer between said fluorophore and saidquencher when said fluorophore is excited; (b) hybridizing said targetbinding sequence to said target sequence, thereby altering saidconformation of said detector nucleic acid, causing a change in afluorescence parameter; and (c) detecting said change in saidfluorescence parameter, thereby detecting said nucleic acid targetsequence.
 2. The method according to claim 1, wherein said detectornucleic acid has a format selected from molecular beacons, scorpionprobes, sunrise probes, conformationally assisted probes and TaqMan™probes.
 3. The method according to claim 1, wherein said detectornucleic acid has an intramolecularly associated secondary structurewhich is a member selected from hairpins, stem-loop structures,pseudoknots, triple helices and conformationally assisted structures. 4.The method according to claim 1, wherein said change in fluorescence isdetected as an indication of the presence of said target sequence. 5.The method according to claim 1, wherein said fluorescence parameter isdetected in-real time.
 6. The method according to claim 1, wherein saiddetector nucleic acid has an intramolecularly base-paired secondarystructure which comprises a portion of said target binding sequence. 7.A method for detecting amplification of a target sequence comprising, inan amplification reaction: (a) hybridizing to said target sequence adetector nucleic acid comprising a single-stranded target bindingsequence and an intramolecularly associated secondary structure 5′ tosaid target binding sequence, wherein at least a portion of saiddetector sequence forms a single stranded tail which is available forhybridization to said target sequence, said detector nucleic acid havinglinked thereto, i) a fluorophore; and ii) a quencher of excited stateenergy having a structure comprising at least three residues selectedfrom aryl, substituted aryl, heteroaryl, substituted heteroaryl andcombinations thereof, wherein at least two of said residues arecovalently linked via an exocyclic diazo bond,  wherein said detectornucleic acid is in a conformation allowing donor-acceptor energytransfer between said fluorophore and said quencher when saidfluorophore is excited; (b) extending said hybridized detector nucleicacid on said target sequence with a polymerase to produce a detectornucleic acid extension product and separating said detector nucleic acidextension product from said target sequence; (c) hybridizing a primer tosaid detector nucleic acid extension product and extending the primerwith said polymerase, thereby linearizing said intramolecularlyassociated secondary structure and producing a change in a fluorescenceparameter; and (d) detecting said change in said fluorescence parameter,thereby detecting said target sequence.
 8. The method according to claim7, wherein said target sequence is amplified by a method selected fromStrand Displacement Amplification, Polymerase Chain reaction 3SR, TMAand NASBA.
 9. The method according to claim 7, wherein said secondarystructure further comprises a partially or entirely single-strandedrestriction endonuclease site.
 10. The method according to claim 7,wherein a change in fluorescence intensity is detected.
 11. The methodaccording to claim 10, wherein said change in fluorescence intensity isdetected in real-time.
 12. The method according to claim 7, wherein saidintramolecularly base-paired secondary structure comprises a portion ofsaid target binding sequence.
 13. A method of ascertaining whether afirst nucleic acid and a second nucleic acid hybridize, said firstnucleic acid comprising a quencher of excited state energy having astructure comprising at least three residues selected from aryl,substituted aryl, heteroaryl, substituted heteroaryl and combinationsthereof, wherein at least two of said residues are covalently linked viaan exocyclic diazo bond, covalently linked to said nucleic acid and asecond nucleic acid, said method comprising: (a) contacting said firstnucleic acid with said second nucleic acid; (b) detecting an alterationin a fluorescent property of a member selected from said first nucleicacid, said second nucleic acid and a combination thereof, therebyascertaining whether said hybridization occurs.
 14. The method accordingto claim 13, wherein said second nucleic acid comprises a fluorophorecovalently attached thereto.
 15. The method according to claim 1,wherein said quencher has a structure according to Formula (I)

wherein R¹, R² and R³ are members independently selected fromsubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl and substituted or unsubstituted unsaturated alkyl, with theproviso that at least two of R¹, R² and R³ are members selected fromsubstituted or unsubstituted aryl and substituted or unsubstitutedheteroaryl; X, Y and Y′ are members independently selected from reactivefunctional groups; f is a number selected from 0 to 4, inclusive, suchthat when (f x s) is greater than 1, the Y′ groups are the same ordifferent; m is a number selected from 1 to 4, inclusive, such that whenm is greater than 1, the X groups are the same or different; n is anumber from 0 to 6, inclusive, such that when (n×t) is greater than 1,the Y groups are the same or different; s is a number from 0 to 6,inclusive, such that when s is greater than 1 the R³ groups are the sameor different; and t is a number from 1 to 6, inclusive, such that when tis greater than 1 the R² groups are the same or different, and when t is1 and s is 0, a member selected from R¹, R² and combinations thereof isa member selected from substituted or unsubstituted polycyclic aryl andsubstituted or unsubstituted polycyclic heteroaryl groups.
 16. Themethod according to claim 1, wherein said quencher has a structureaccording to Formula (II)

wherein X and Y are members independently selected from reactivefunctional groups; m is a number selected from 1 to 5, inclusive, suchthat when m is greater than 1, the X groups are the same or different; nis a number selected from 0 to 5, inclusive, such that when m is greaterthan 1, the Y groups are the same or different; s is a number selectedfrom 1 to 5, inclusive, such that when s is greater than 1, the R³groups are the same or different; R¹, R², and R³ are membersindependently selected from substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, and substituted orunsubstituted unsaturated alkyl, with the proviso that at least two ofR¹, R² and R³ are members independently selected from substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl.
 17. Themethod according to claim 16, wherein R² and R³ are membersindependently selected from aryl and aryl substituted with a memberselected from amino, amino derivatives, nitro, C₁-C₆ alkyl, C₁-C₆ alkoxyand combinations thereof; R¹ includes a structure according to FormulaIII:

wherein R⁴ is a member selected from alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl and substituted heteroaryl.
 18. The methodaccording to claim 1, wherein said quencher has a structure according toFormula IV:

wherein X and Y are members independently selected from reactivefunctional groups; m is a number selected from 0 to 4, inclusive, suchthat when m is greater than 1, the X groups are the same or different; cis a number selected from 0 to 4, inclusive, such that when c is greaterthan 1, the R³ groups are the same or different; v is a number from 1 to10 and when v is greater than 1, the R² groups are the same ordifferent; R¹, R², and R³ are members independently selected fromsubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl and substituted or unsubstituted unsaturated alkyl, with theproviso that at least two of R¹, R² and R³ are members selected fromsubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl and combinations thereof.
 19. The method according to claim1, wherein said quencher has a structure according to Formula V:

wherein R⁵, R⁶ and R⁷ are members independently selected from —NR′R″,substituted or unsubstituted aryl, nitro, substituted or unsubstitutedC₁-C₆ alkyl, and substituted or unsubstituted C₁-C₆ alkoxy, wherein R′and R″ are independently selected from H and substituted orunsubstituted C₁-C₆ alkyl; X and Y are independently selected from thegroup consisting of reactive functional groups; m is a number from 1 to2, inclusive, such that when m is 2, the X groups are the same ordifferent; n is a number from 0 to 1, inclusive; a is a number from 0 to4, inclusive, such that when a is greater than 1, the R⁵ groups are thesame or different; b is a number from 0 to 4, inclusive, such that when(v×b) is greater than 1, the R⁶ groups are the same or different; c is anumber from 0 to 5, inclusive, such that when c is greater than 1, theR⁷ groups are the same or different; and v is a number from 1 to 10,inclusive, such that when v is greater than 1, the value of b on each ofthe b phenyl rings is the same or different.
 20. The method according toclaim 1, wherein said quencher has a structure according to Formula VI:

wherein R⁵, R⁶ and R⁷ are members independently selected from amine,alkyl amine, substituted or unsubstituted aryl, nitro, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted C₁-C₆ alkoxy; ais a number between 0 and 5, inclusive, such that when a is greater than1, the R⁵ groups are the same or different; b is a number between 0 and4, inclusive, such that when b is greater than 1, the R⁶ groups are thesame or different; c is a number between 0 and 4, inclusive, such thatwhen c is greater than 1, the R⁷ groups are the same or different; andX¹ and X² are members independently selected from C₁-C₆ alkyl or C₁-C₆substituted alkyl, —OH, —COOH, —NR′R″, —SH, —OP(OX³)(NR′R″) wherein R′and R″ are members independently selected from the group consisting ofH, and alkyl or substituted alkyl.
 21. The method according to claim 1,wherein said quencher has a structure which is a member selected from:

wherein X⁵ and X⁶ are members independently selected from H, substitutedor unsubstituted C₁-C₆ alkyl, —OR′, —COOR′, —NR′R″, —SH, —OP(OX³)N(X⁴)₂,in which R′ and R″ are members independently selected from the groupconsisting of H, and alkyl or substituted alkyl, with the proviso thatat least one of X⁵ and X⁶ is a reactive functional group wherein X³ andX⁴ are members independently selected from CN and substituted orunsubstituted C₁-C₆ alkyl.
 22. The method according to claim 1, whereinsaid nucleic acid target sequence is in a mixture.
 23. The methodaccording to claim 1, wherein said nucleic acid target sequence is partof a mixture, and wherein said mixture further comprises a seconddetector nucleic acid comprising a second single-stranded target bindingsequence, wherein said second detector nucleic acid hybridizes to asecond target sequence, said second detector nucleic acid having linkedthereto, i) a second fluorophore; and ii) a second quencher of excitedstate energy; wherein said second detector nucleic acid is in aconformation allowing donor-acceptor energy transfer between said secondfluorophore and said second quencher when said fluorophore is excited.24. The method according to claims 1, 2, 3, 7 and 23, wherein saiddetector nucleic acid is an oligonucleotide.